Compositions and methods for modifying toxic effects of proteinaceous compounds

ABSTRACT

The present invention provides methods to produce immunotoxins (ITs) and cytokines with a reduced ability to promote vascular leak syndrome (VLS). The invention also provides ITs and cytokines which have been mutated to lack amino acid sequences which induce VLS. Also disclosed are methods for producing peptides that inhibit the induction of VLS by ITs and cytokines. Also disclosed are peptides comprising the (x)D(y) sequence to promote the extravasation of other molecules. Toxins mutated in the (x)D(y) motif or active site residues are disclosed for used in vaccines.

This application is a continuation of U.S. patent application Ser. No.09/538,873, filed Mar. 30, 2000, now U.S. Pat. No. 6,566,500, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/126,826,filed Mar. 30, 1999, the disclosure of which is specificallyincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of vascular leak,and particularly concerns toxins which induce or cause vascular leaksyndrome (VLS). The invention provides immunotoxins (ITs) and cytokineswhich have been mutated to lack amino acid sequences which induce VLSand other toxic side effects. Disclosed are methods for mutating DNAsegments encoding cytokines or immunotoxins so that an immunotoxin isproduced that lacks sequences that induce VLS and other toxic sideeffects. Also disclosed are methods of preparing and using peptideswhich promote VLS and thus aid delivery of molecules into tissues. Thepresent invention also relates to methods of preparing peptides whichinhibit VLS and the used of mutated toxins as vaccines to protectimmunized individuals from later toxicity.

2. Description of Related Art

VLS is often observed during bacterial sepsis and may involve IL-2 and avariety of other cytokines (Baluna and Vitetta, 1996). The mechanismsunderlying VLS are unclear and are likely to involve a cascade of eventswhich are initiated in endothelial cells (ECs) and involve inflammatorycascades and cytokines (Engert et al., 1997). VLS has a complex etiologyinvolving damage to vascular endothelial cells (ECs) and extravasationof fluids and proteins resulting in interstitial edema, weight gain and,in its most severe form, kidney damage, aphasia, and pulmonary edema(Sausville and Vitetta, 1997; Baluna and Vitetta, 1996; Engert et al.,1997). Vascular leak syndrome (VLS) has been a major problem with allITs thus far tested in humans, as well as cytokines such as interleukin2 (IL-2), TNF and adenovirus vectors (Rosenberg et al., 1987; Rosenstenet al., 1986).

ITs are hybrid molecules consisting of monoclonal antibodies (MAbs) orother cell-binding ligands, which are biochemically or geneticallylinked to toxins, toxin subunits, or ribosome inactivating proteins(RIPs) from plants, fungi or bacteria (Vitetta et al., 1993). Over thepast two decades, ITs containing deglycosylated (dg) ricin A chain(dgRTA) have been developed, structurally optimized for stability andactivity and evaluated for activity both in vitro, and in vivo inrodents, monkeys and humans (Vitetta et al., 1993; Sausville andVitetta, 1997; Baluna and Vitetta, 1996).

It has been postulated that dgRTA-ITs induces VLS by damaging vascularendothelial cells (Soler-Rodriguez et al., 1993; Baluna et al., 1996).IL-2 and ITs prepared with the catalytic A chain of the plant toxin,ricin (RTA) and other toxins, damage human ECs in vitro and in vivo(Dutcher et al., 1991; Rosenberg et al., 1987; Vial and Descotes, 1992).Studies using human umbilical vein ECs (HUVECs) demonstrated that dgRTAor ITs prepared with dgRTA can damage these cells within one hour(Soler-Rodriguez et al., 1993) while the inhibition of protein synthesisrequired 4 hrs or longer. DgRTA-ITs also interfere with fibronectin(Fn)-mediated adhesion (Baluna et al., 1996). Fn inhibits dgRTA-mediateddamage to human umbilical vein endothelial cells (HUVECS) (Baluna etal., 1996). Cell adhesion to Fn is mediated by integrins which recognizeRGD and LDV sequences in the Fn molecule (Makarem and Humphries, 1991;Wayner and Kovach, 1992).

Three MAbs linked to dgRTA have been evaluated in Phase I trials in over200 patients with relapsed chemorefractory lymphoma, myeloma, Hodginsdisease and graft vs. host disease (GVHD) (Sausville and Vitetta, 1997).These ITs have shown no evidence of myelotoxicity or hepatotoxicity, butall have induced VLS at the maximum tolerated dose (MTD) as defined byhypoalbuminemia, weight gain, and in the most severe cases, pulmonaryedema and hypotension (Baluna et al., 1996). In addition, they haveinduced myalgia and, in 3% of patients, rhabdomyalyosis at the MTD(Sausville and Vitetta, 1997); this side effect may also be related toVLS and result from muscle edema. Further, aphesias have occurred in <5%of patients' these may be due to edema in the cerebral microvasculture.

Despite this dose limiting toxicity (DLT) clinical responses usingdgRTA-ITs have been encouraging with 15-30% of chemorefractory relapsedlymphoma patients experiencing objective partial or complete response inPhase I clinical trials (Sausville and Vitetta, 1997). However, the DLT,VLS, has decreased the enthusiasm for continuing on to Phase II and IIItrials in patients.

Clearly, further development of dgRTA-ITs as well as other ITscontaining toxins and RIPS, as well as cytokines as clinical agentswould be greatly facilitated by the elimination or reduction of VLS. IfVLS could be avoided or reduced it would permit the use of much higherdoses of a variety of therapeutic agents such as Its, gene therapy andcytokines without the dose limiting side effects currently encountered.

SUMMARY OF THE INVENTION

The present invention overcomes the deficiencies in the art by providingmethods for modulating the ability of various proteinacious compounds toinduce toxic effects, and proteinacious compounds that have beenmodified such that they have modulated ability to induce toxic effects.In some embodiments, the invention allows for the production of ITs witha reduced ability to promote or induce such toxic effects, including,for example, VLS. ITs made in accordance with the invention are for anynumber of therapeutic applications, for example, the treatment of GVHD,non-Hodgkin's and Hodgkin's lymphoma, myloma, and metastatic lesionstumors, in some particular aspects solid tumors. The present inventionalso provides methods for reducing the VLS promoting ability ofproteinaceous compositions through a mutation of sequences that induceor promote any of a number of toxic effects. The present inventionprovides ITs, IL-2 TNF and adenovirus with a reduced ability to promotetoxic effects, and methods of using such compounds.

The invention, in one aspect, provides a method of modifying the abilityof a proteinaceous composition to induce a toxic effect, comprising thesteps of: identifying at least one amino acid sequence comprising thesequence (x)D(y), wherein (x) is selected from the group leucine,isoleucine, glycine and valine, and wherein (y) is selected from thegroup valine, leucine and serine; and altering the amino acid sequencecomprising the sequence (x)D(y). In certain embodiments, the alteringcomprises at least one mutation of the amino acid sequence. In otherembodiments, the amino acid sequence is removed. In particular aspects,the amino acid sequence comprises the sequence (x)D(y), wherein the(x)D(y) sequence is GDL, GDS, GDV, IDL, IDS, IDV, LDL, LDS, LDV, LDS,VDL or VDV. In certain more specific embodiments, the invention providesa modified proteinaceous composition that has altered, relative to thesequence of a native proteinaceous composition, at least one amino acidof a sequence comprising (x)D(y), wherein (x) is selected from the groupleucine, isoleucine, glycine and valine, and wherein (y) is selectedfrom the group valine, leucine and serine, for use as a medicament.

In certain aspects, the composition has a reduced ability to induce atleast one toxic effect. In other aspects, the altering enhances theability of the composition to induce at least one toxic effect. Inparticular embodiments, the toxic effect is, for example, VLS, theability to induce apoptosis, a disintigrin-like activity, the ability todamage EC cells or a combination thereof. Of course, those of ordinaryskill will, by following the teachings of this specification, be able todetermine additional toxic effect that may be modulated according to themethods disclosed herein. In some embodiments of the invention, it isdesirable to decrease the level of the toxic effect. For example, thereis great benefit to be gained by creating an IT which exhibits no, orlessened, VLS. In alternative embodiments, it will be desirable toincrease the level of a given toxic effect. For example, if aproteinacious compound is being used to induce apoptosis in atherapeutic manner, then modifying the compound to increase the level ofapoptosis could be beneficial. Those of ordinary skill in the art willbe able to determine any of a number of manners in which to employ themodulation of toxic effects that can be had with the methods of theinvention.

In some embodiments, the (x)D(y) sequence comprises a residue on thesurface of the composition. In certain aspects, the altering occurs atone or more (x)D(y) tri-amino acid sequences. In certain embodiments,the (x)D(y) sequence comprises at least one flanking sequence. Inparticular aspects, the altering the sequence comprises at least onealteration within the at least one flanking sequence. In certainaspects, the at least one flanking sequence is mutated. In some facets,the at least one flanking sequence is removed. In other facets, thealteration occurs within of from about 1 to about 6 residues of an (x)or an (y) of a (x)D(y) tripeptide sequence. In some aspects, theflanking sequence is C-terminal to the (x)D(y) sequence. In otheraspects, the flanking sequence is N-terminal to the (x)D(y) sequence. Ina particular facet, the at least one flanking sequence comprises twoflanking sequences, wherein the two flanking sequences are N-terminaland C-terminal to the (x)D(y) sequence.

The proteinacious composition can be any presently known of discoveredin the future that has the (x)D(y) tripeptide sequence. In someembodiments, the proteinaceous composition comprises a toxin, acytokine, a viral sequence or a combination thereof. In particularaspects, the toxin comprises a plant toxin, a fungal toxin, a bacterialtoxin, a RIP or a combination thereof. In certain facets, the toxincomprises Abrin A chain, Diphtheria Toxin (DT) A-Chain, Pseudomonasexotoxin, RTA, Shiga Toxin A chain, Gelonin, Momordin, PokeweedAntiviral Protein, Saporin, Trichosanthin, Barley toxin or a combinationthereof. In other embodiments, the proteinaceous composition comprises acytokine, such as, for example, Interleukin-2. In further embodiments,the proteinaceous composition comprises a viral sequence, such as, forexample, an adenoviral sequence. In certain aspects, the proteinaceouscomposition further comprises, or is comprised in, an IT.

The invention, in some particularly preferred aspects, the inventionprovides a method of reducing the ability of a proteinaceous compositionto promote VLS, comprising the steps of: identifying at least one aminoacid sequence comprising the sequence (x)D(y), as defined above.

In certain aspects, the invention provides the use of a modifiedproteinaceous composition that has altered, relative to the sequence ofa native proteinaceous composition, at least one amino acid of asequence comprising (x)D(y), for the manufacture of a medicament for thetreatment of a disease, including but not limited to GVHD, non-Hodgkin'sand Hodgkin's lumphoma, myeloma, as well as metastatic lesions of solidtumors and damage to endothelial cells (i.e., VLS).

The invention additionally provides a method of preparing an IT with areduced ability to induce a toxic effect, comprising the steps of:identifying at least one amino acid sequence comprising the sequence(x)D(y); removing the amino acid sequence from the toxin; andconjugating the toxin to a composition comprising at least one antibodyto produce an IT, wherein the IT produced possesses a reduced ability topromote a toxic effect when compared to a like IT wherein the amino acidsequence was not removed from the toxin.

The invention also provides a method of enhancing the ability of aproteinaceous composition to induce extravasation, comprising adding atleast one amino acid sequence comprising (x)D(y) to the composition. Inparticular aspects, the composition comprises a peptide. In furtheraspects, the extravasation of the composition or at least one moleculeis enhanced. In some embodiments, the composition is covalentlyconjugated to the at least one molecule. In certain facets, theextravasation of the molecule is enhanced. In additional facets, themolecule is a therapeutic agent, such as, for example, at least one IT,antibody, cytokine, virus or a combination thereof.

The invention further provides a method of reducing the toxic effects ofa proteinaceous material in a patient, comprising administering to apatient a composition that mimics a sequence comprising (x)D(y). Inparticular aspects, the proteinaceous material comprises at least one(x)D(y) sequence. In additional aspects, the composition comprises atleast one amino acid sequence comprising the sequence (x)D(y), whereinthe amino acid sequence has been altered to possess an reduced abilityto promote a toxic effect. In certain facets, the composition comprisesat least one peptide.

The invention also provides a modified proteinaceous composition thathas altered, relative to the sequence of a native proteinaceouscomposition, at least one amino acid of a sequence comprising (x)D(y),prepared according to the methods described above and elsewhere in thisspecification. In certain embodiments, the proteinaceous compositioncomprises a toxin, a cytokine, a viral sequence or a combinationthereof. In certain aspects, the toxin is, for example, a plant toxin, afungal toxin, a bacterial toxin, a RIP or a combination thereof. Inadditional aspects, the toxin comprises Abrin A chain, Diphtheria Toxin(DT) A-Chain, Pseudomonas exotoxin, RTA, Shiga Toxin A chain, Gelonin,Momordin, Pokeweed Antiviral Protein, Saporin, Trichosanthin, Barleytoxin or a combination thereof. In other embodiments, the proteinaceouscomposition comprises a cytokine, such as for example, Interleukin-2. Inother aspects, the proteinaceous composition comprises a viral sequence,such as, for example, an adenoviral sequence.

In certain facets, the composition further comprises an antibody. Inparticular aspects, the composition further comprises an IT. Inadditional facets, the IT further comprises at least a second agent,such as, for example, at least one effector molecule. In particularaspects, the effector molecule is a toxin, an anti-tumor agent, atherapeutic enzyme, an antiviral agent, a virus, a cytokine, a growthfactor, or a combination thereof. In other facets, the agent is at leastone reporter molecule.

The invention additionally provides an IT, comprising at least oneproteinaceous molecule with a reduced ability to induce VLS, apoptosis,disintegrin-like activity or EC damage, wherein the proteinaceousmolecule has at least one (x)D(y) or flanking sequence altered.

The invention provides a modified proteinaceous composition with anenhanced ability to promote extravasation, wherein the compositioncomprises at least one amino acid sequence comprising a (x)D(y)tripeptide or a flanking sequence relative to the native sequence.

In certain embodiments, the composition comprises a therapeutic agent,such as, for example, at least one IT, antibody, cytokine, virus or acombination thereof. In specific embodiments, the composition and thetherapeutic agent are covalently conjugated. In particular facets, thecomposition is a therapeutic agent.

The invention also provides a RTA with a reduced ability to promotetoxicity in a patient, wherein the (x)D(y) sequence comprising positions74 to 76 is altered. In certain embodiments, the leucine at position 74is altered, the aspartate at position 75 is altered, and/or the valineat position 76 is altered. In specific facets, the (x)D(y) sequencefurther comprises positions of from about 1 to about 6 residues of an(x) or an (y) of the (x)D(y) tripeptide sequence.

The invention also provides a method of reducing the ability of aproteinaceous composition to induce VLS, comprising the steps of:identifying at least one amino acid sequence (x)D(y); and altering,removing or mutating the amino acid sequence.

Flanking regions to the (x)D(y) tri-peptide sequence may be altered toreduce a proteinaceous composition's ability to induce VLS. As usedherein, a “proteinaceous composition” refers to a protein of greaterthan about 200 amino acids or the full length endogenous sequencetranslated from a gene, a polypeptide of greater than about 100 aminoacids, and/or a peptide of from about 3 to about 100 amino acids,including peptides of 3, 4, 5, 6, etc., 10, 11, 12, 13, 14, etc., 20,21, 22, etc, 30, 40, 50, 60, etc. 100, 110, 120, etc. 200, 220, 240,etc, 300, 350, 400, etc, 500, 600, 700, etc., and 1000 amino acids inlength. In an aspect the C-terminal flanking amino acid sequence may bealtered, mutated or removed if it is a threonine (T). In certainaspects, this method of altering this sequence and/or flanking aminoacids is by removal of the amino acid sequence.

The invention provides a proteinaceous composition that has a reducedability to induce VLS. In an aspect, the proteinaceous composition thathas been altered to remove at least one amino acid sequence contiguouswith the composition comprising the sequence (x)D(y). In certain aspectsof the present invention, the proteinaceous composition may be aribosome-inactivating protein (RIP), including but not limited togelonin, momordin, pokeweed antiviral protein (PAP), saporin, ortrichosanthin; a toxin or toxin subunit, including but not limited toabrin A chain, diphtheria toxin (DT) A-chain, Pseudomonas exotoxin-A(PE38-lys), RTA, Shiga toxin A chain, or barley toxin; a cytokineincluding but not limited to IL-2. Proteins, polypeptides and/orpeptides may be derived from RIPs, toxins or cytokines to be used in themethods and compositions of the present invention. In certain aspects,the proteinaceous composition may be used to make an IT with a reducedability to promote or enhance VLS. In other aspects, the proteinaceouscomposition for use in an IT is a RIP and/or toxin sequence.

In other aspects of the present invention, proteins, peptides and/orpolypeptides may be made that include the VLS-inducing sequence. TheseVLS-inducing proteinaceous compositions may be used to promote VLS, andincrease the extravasation of molecules into tissues. In additionalaspects of the present invention, proteinaceous compositions may be madethat lack the VLS-inducing sequence. Such compositions may be used asinhibitors of agents that induce VLS in vivo or adenoviral vectors forgene therapy. The compositions of the present invention may be made bysynthetic peptide synthesis or through the use of recombinant genetictechnology, as would be known to those of ordinary skill in the art inlight of the present disclosure. Toxin mutants lacking VLS activity canalso be effective for protecting individuals against the native toxin.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A and 1B. The in vivo effect of RFB4-RTA-peptides. (FIG. 1A) SCIDmice with vascularized human skin xenografts were injected with 200 μgof RFB4-dgRTA (solid), RFB4-LDV⁺ (open), RFB4-GQT (cross-hatched) orsaline (hatched), and the wet/dry weight ratios of biopsies of the humanskin were determined. (FIG. 1B) SCID mice were injected as described inFIG. 1A and the wet/dry weight ratios of lungs were determined. Thevalues represent the mean of three experiments±SD. The asterisksindicate a statistically significant difference from saline (−) treatedmice (*, p<0.02, **p<0.01).

FIGS. 2A and 2B. Inhibition of the binding of dgRTA and RFB4-LDV+ toHUVECs. (FIG. 2A) 10⁵ HUVECs were incubated on ice for 30 min withFITC-dgRTA, in the presence or absence of 100-fold excess of dgRTA(solid), RFB4-LDV⁺ (crosshatched), RFB4 (shaded), Fn (hatched) orPE38-lys (open) in 100 ul PBS/BSA/Azide. The percent inhibition ofbinding to HUVECs is presented. The values represent the means±SD ofthree studies. (FIG. 2B) The same as FIG. 2A, except the 10⁵ HUVECs wereincubated on ice for 30 min with FITC-RFB4-LDV⁺.

FIG. 3. RFB4-rRTA ITs IC₅₀ determinations. Selected examples of LD₅₀determinations by in vitro cytotoxicity assays, where IC₅₀ is calculatedas the concentration of IT at which [³H] leucine incorporation wasinhibited by 50% relative to untreated control Daudi cell culture.

FIG. 4. Effect of RFB4-rRTA ITs on the morphology of HUBEC monolayers.HUVEC monolayers were incubated at 37° C. for 18 h with 100 μg/ml ofRFB4rRTA ITs in M199 medium with 2% fetal calf serum. Morphologicalchanges were scored as: −, no changes; rounding up of cells; and ++disruption and detachment of cells from the monolayer. The toxicitygrade was represented as a ratio (number of “+”/number of experiments).

FIGS. 5A and 5B. In vivo effect of RFB4-rRTA ITs. SCID mice wereinjected with 200 μg of RFB4-rRTA ITs of saline. (FIG. 5A) The bodyweights were determined. (FIG. 5B) The wt/dry wt ratio of lungs weredetermined.

FIG. 6. Profile of Acid-Treated Sepharose 4B Column-Purification ofRicin

FIG. 7. Profile of Sephacryl S-200 Column-Separation of RCA-1 and RCA-2.

FIG. 8. Profile of DEAE Sepharose Column and Acid-Treated Sepharose 4BColumn-Separation of dgRTA and DGRTB Chains.

FIG. 9. Profile of Blue-Sepharose CL-4B Column—Purification of dgRTA.

FIG. 10. Profile of Asialofetulin-Sepharose Column—Purification ofdgRTA.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Cell damage, particularly endothelial cell damage, whether produced bytoxins, such as from snake bites or molecules causing septic shock, ortherapeutic agents, such as ITs or interleukins, remains a problem forpatients. Types of cell damage include VLS, disintigrin-like activitiesand apoptosis.

To devise methods and compositions to alleviate VLS, candidate sequencesof molecules which cause VLS were evaluated to determine whether RTA,toxins, RIPS and IL-2 might share structural motifs responsible forinterfering with cell-cell and cell-matrix interactions, and therebydamage human ECs. In comparing the sequences of VLS-inducing toxins,RIPS and IL-2, a (x)D(y) consensus motif was identified where (x) couldbe L, I, G or V and (y) could be V, L or S. In the case of RTA and IL-2,molecular modeling indicated that these motifs were completely orsubstantially exposed on the surface of their respective molecules. Asimilar motif is shared by viral disintegrins, which disrupts thefunction of integrins, indicating that RTA, IL-2 and perhaps othertoxins may damage ECs by virtue of their (x)D(y) motifs and hence, maybe disintegrins.

This vascular-leak promoting activity of this motif was surprising andunexpected, since LDV homologue sequences also play a role in thevascular functions of a variety of non-toxic molecules includingvascular cell adhesion molecule 1 (VCAM-1), which contains the IDSsequence, and the γ chain of fibrinogen, which contains the GDV sequence(Clements et al., 1994). LDV constitutes the minimal active site in theCS1 domain of fibronectin responsible for its binding to the α₄β₁integrin receptor (Makarem and Humphries, 1991; Wayner and Kovach, 1992;Nowlin et al., 1993). Though fibronectin possesses this sequence, itdoes not damage HUVECs. Instead, FN protects HUVECs from RTA-mediateddamage (Baluna et al., 1996), in direct contrast to the VLS activity oftoxic agents that possess this motif.

To determine whether this motif was responsible for EC damage, short LDVor LDL containing peptides from RTA or IL-2, respectively, weregenerated, attached to a mouse MAb and studied their ability to bind toand damage HUVECs in vitro and to damage mouse lung vasculature andhuman vasculature in skin xenografts in vivo. One active site mutant ofRTA and several LDV mutants were generated. These LDV mutants containedconservative changes which, when modeled, would not be expected toaffect the active site of the RTA. Antibody-conjugated peptides from RTAcontaining the sequence (L74, D75, V76), but not peptides with deletedor altered sequence, induced EC damage in vitro and vascular damage invivo in the two animal models (Baluna et al., 1999). These resultsdemonstrated that the VLS-inducing site does not require the activesite. It is contemplated that the noncontiguous active site of the RTA,which does not encompass LDV, is either not required to damage ECs, oronly partly contributes to vascular damage.

These results demonstrate that an active site may not be required toinduce vascular damage, and that one or more active peptides orpolypeptides may be made with reduced VLS promoting activity. With thisdiscovery, it is now possible that one or more amino acid deletion(s) ormutation(s) of the (x)D(y) sequence(s), and/or at least one regionflanking the sequence, may reduce or prevent VLS and improve thetherapeutic index or the tolerated dose of VLS-inducing molecules. It isexpected that one or more peptides and small molecule drug inhibitorscomprising at least one mutated motif and/or one or more flankingsequence can be created that reduce or eliminate the VLS induced by VLSpromoting agents.

In certain embodiments, it is contemplated that disintegrin ordisintegrin-like activity of proteinaceous compositions may be reducedor enhanced. Disintegrins possess various activitie(s) including anability to damage ECs, an ability to interfere with cell adherenceand/or an ability to interfere with platelet aggregation. It iscontemplated that one or more amino acid deletion(s) or mutation(s) ofthe (x)D(y) sequence(s), and/or one or more flanking residues, mayreduce or prevent the disintegrin-like activity of one or more moleculescomprising these sequences. It is expected that one or more peptides andsmall molecule drugs inhibitors comprising at least one mutated motifand/or one or more flanking sequence can be created that reduce oreliminate the disintegrin-like activity of such agents.

Additionally, the LDV site of RTA induced apoptosis in ECs. It has beenreported that many toxins and ITs induce apoptosis as well as inhibitprotein synthesis. It is also contemplated that one or more amino aciddeletion(s) or mutation(s) of the (x)D(y) sequence(s), and/or at leastone flanking region, may reduce or prevent the apoptotic activity of oneor more molecules comprising these sequences. It is expected that one ormore peptides and small molecule drug inhibitors comprising at least onemutated motif and/or at least one flanking sequence can be created thatreduce or eliminate the apoptotic activity of such agents. Thusapoptotic activity may cause or contribute to toxin or cytokine inducedVLS.

It is also contemplated that one or more amino acid deletion(s) ormutation(s) of the (x)D(y) sequence(s), and/or one or more flankingresidues, may reduce or prevent the ability of molecules comprisingthese sequences to induce EC damage. It is expected that one or morepeptides and small molecule drug inhibitors comprising at least onemutated motif and/or one ore more flanking residuces can be created thatreduce or eliminate the EC damaging activity of such agents.

Described herein below are methods and compositions with reduced orenhanced VLS promoting abilities based upon mutations in the (x)D(y) or(x)D(y)T sequences within proteins, polypeptides, peptides or otherproteinaceous materials which remove or add such sequences,respectively. It is contemplated that the same mutations described forreducing or enhancing VLS promoting ability will also reduce or enhance,respectively, the apoptotic activity, EC damaging and/or one or moredisintegrin-like activities of polypeptides, peptides or proteins. Thus,it will be understood that all methods described herein for producingproteins, polypeptides and peptides with enhanced or reduced VLSpromoting ability will be applied to produce proteins, polypeptides andpeptides with reduced apoptotic activity, EC damaging and/or one or moredisintegrin-like activities. All such methods, and compositionsidentified or produced by such methods, are encompassed by the presentinvention.

A. Identification of an (X)D(Y) Motif in VLS-Inducing Agents

Homologous structural motifs in RTA, other toxins, RIPs and IL-2, whichmay affect cell-cell and cell-matrix interactions and thereby damagehuman ECs, have been identified and tested for their ability to promoteVLS in model systems. The (x)D(y) motif where x=L, I, G or V and y=V, Lor S (Table 1) is common in the sequences of RTA, other toxins, RIPs andcytokines which induce VLS. This motif is also shared by viraldisintegrins which disrupt the function of integrins (Coulson et al.,1997).

TABLE 1 Non-Limiting Examples of (x)D(y) Motifs in Molecules WhichInduce VLS GenBank or (X) D(Y) GenPept Category Agent inducing VLS MotifLocation Accession # Toxins¹ Abrin A chain IDV 68-70 X76721 GDL 114-116VDS 229-231 Barley toxin LDV 171-173 U77463 Diphtheria Toxin VDS 6-8576189 (DT) A-Chain VDS 28-30 IDS 289-291 GDV 430-432 GDL 605-607 RicinToxin LDV 74-76 A23903 A-Chain (RTA) Shiga toxin A chain VDS 36-38M19437 IDS 63-65 VDV 74-76 GDS 132-134 LDL 162-164 VDL 219-221 RIPs³Gelonin IDV 114-116 L12243 Momordin LDV 64-66 576194 LDS 132-134Momordin LDS 165-167 P16094 Pokeweed Antiviral VDS 179-181 X98079Protein (PAP) GDL 308-310 Saporin LDL 6-8 X69132 IDL 143-145Trichosanthin GDV 23-25 U25675 IDV 87-89 LDS 155-157 CytokinesInterleukin-2 (IL-2) LDL 19-21 1311005 ¹The enzymatically active chainof the holotoxin ²PE38 refers to enzymatically active Domain III(residues 405 to 613) plus residues 253-354 and 381-404 in PE.³Ribosome-inactivating proteins (RIPs) which are homologues of theenzymatically active A chains of plant toxins

1. Localization of (x)D(y) Motifs in RTA, Disintegrins, PE38-lys andIL-2

With the discovery of the importance of the (x)D(y) sequence inpromoting VLS, it is now possible to create RTA mutants which willretain their enzymatic activity, which is important for making effectiveITs, but which also have their VLS-inducing properties reduced.

The LDV motif in RTA (residues 74-76, SEQ ID NO:1) is at the C-terminusof a β-strand of the first domain near the Tyr-80 residue which isinvolved in the active site (Mlsna et al., 1993). The active site(residues 80, 123, 177, 180, 211) of the enzyme does not include the LDVsequence so that the enzymatic activity of RTA should not be affected bymutations or deletions in this sequence (Munishkin and Wool, 1995).

To examine the crystal structure of RTA and IL-2, space filling modelsof the three dimensional structures of RTA (PDB accession number1br5.pdb) and IL-2 (PDB accession number 1ir1.pdb) were compared withthe atoms of the LDV residues of RTA, the LDL residues of IL-2, and theactive site residues of RTA (Y80, Y123, E177, R180, N209 and W211). Themodels were generated with the Insight II program (MSI). Examinations ofthe crystal structure of RTA indicate that this motif is only partiallyexposed, but structural fluctuations in the molecule may increase itsaccessibility. From this and other data described herein, it iscontemplated that either alterations in the (x)D(y) motif, theC-terminal flanking amino acid(s), the N-terminal flanking aminoacid(s), or a combination thereof, may result in the loss ofVLS-inducing activity by a variety of agents.

Another family of proteins called disintegrins usually contain an RGDsequence. In the case of one disintegrin, which is present in rotavirus,an LDV sequence is present (Coulson et al., 1997). Disintegrins damageECs or interfere with cell adherence and/or platelet aggregation (McLaneet al., 1998; Huang, 1998; Tselepis et al., 1997). In the snake venomdisintegrin kistrin, LDV can be substituted for RGD without compromisingdisintegrin function (Tselepis et al., 1997). Thus, RTA and a variety ofother molecules may be disintegrins which share properties with kirstin(Blobel and White, 1992; Lazarus and McDowell, 1993) in damaging humanECs. In certain embodiments, it is contemplated that disintegrins ormolecules that possess disintegrin-like activity may be altered orproduced to possess a reduced ability to damage ECs, a reduced abilityto interfere with cell adherence and/or a reduced ability to interferewith platelet aggregation. Such molecules may be produced by mutating atleast one residue in the (x)D(y) sequence or at least one flankingresidue. It is also contemplated that peptides or peptide mimics of the(x)D(y) and/or flanking sequences may be made that block the activity ofdisintegrins.

In PE38-lys the GDL sequence is distal from the active site (Li et al.,1995). Thus, it is contemplated that PE38-lys may be similarly mutatedto reduce or eliminate its VLS promoting activity without completelyeliminating its activity.

In IL-2, the LDL sequence at residues 19-21 (SEQ ID NO:2) is located inan α-helix and is also partially exposed. A mutation in Asp-20, in theLDL motif (Table 1) eliminates binding of IL-2 to the β chain of theIL-2 receptor and subsequent cell proliferation (Collins et al., 1988).It has been reported that IL-2 directly increases the permeability ofthe vascular endothelium to albumin in vitro and that this effect can beinhibited by anti-IL-2 receptor MAbs (Downie et al., 1992). The resultsof Example 1 demonstrate that the LDL sequence in IL-2 damages HUVECs.However, in contrast to RTA, the Asp-20 in the LDL of IL-2 is involvedin receptor binding and functional activity (Collins et al., 1988).Thus, it is contemplated that in certain embodiments, mutations inIL-2's (x)D(y) sequence and/or flanking sequence(s) to eliminate orreduce VLS must preserve the Asp-20 or the biological activity of IL-2may be reduced.

2. Mutations in Flanking Sequences

The (x)D(y) sequence may not be solely responsible for the promotion ofVLS. In certain embodiments, it is contemplated that additionalsequences that flank the (x)D(y) sequence may be mutated to enhance orreduce a peptide, polypeptide or protein's ability to promote VLS.

For example, LDV constitutes the minimal active site in the CS1 domainof fibronectin responsible for its binding to the α₄β₁ integrin receptor(Makarem and Humphries, 1991; Wayner and Kovach, 1992; Nowlin et al.,1993). However, fibronectin (FN) does not damage HUVECs. Instead, FNprotects HUVECs from RTA-mediated damage (Baluna et al., 1996). UnlikeRTA, FN has a C-terminal LDV-flanking proline instead of a threonine.

In disintegrins, residues flanking RGD, play a role in ligand binding(Lu et al., 1996). The difference between the ability of an LDV orhomologue-containing molecule to promote vascular integrity (e.g., FN)or disrupt it (e.g., RTA) may depend on the orientation, or availabilityfor interaction (i.e., binding), of the LDV motif and hence, on flankingsequences. Therefore, a change in one or more amino acids of thissequence or one or more amino acids of the N- or C-terminal flankingsequences may convert a molecule from one that damages endothelial cells(distintegrin-like) to one that enhances their growth. It iscontemplated that changes in one or more flanking residues of the(x)D(y) sequence may enhance or reduce the ability of a molecule topromote VLS. It is further contemplated that changes that expose the(x)D(y) sequence to the external surface of the protein so as tointeract with other proteins, such as receptors, would enhance VLSpromoting activity, while conformations that are less exposed may reduceVLS promoting activity.

B. Production of Compositions with Altered VLS Activity

With the identification of the (x)D(y) and the (x)D(y)T motifs asinducing VLS, inducing apoptosis, and other effects, it is possible thatthe creation of a new family of molecules of VLS inhibitors will allowthese molecules to exert maximal beneficial effects. For example, areduced toxicity of anti-cancer therapeutic agents using thecompositions and methods disclosed herein may allow larger tumors ormore advanced disease to be treated. It is now possible to identify orsynthesize small drug molecule(s) which block the interaction betweencells and VLS promoting, apoptosis promoting, EC damaging, and/ordistintegrin-like molecules. In certain embodiments, peptides ordrug-mimetics based on the (x)D(y) and/or (x)D(y)T motif or its flankingsequences may be used to inhibit VLS or other activities in vivo. It ispossible to create peptides or peptide-carrier conjugates which competewith the LDV motif binding site on endothelial cells and prevent VLS orother actions in a variety of other situations including sepsis, IL-2therapy, etc.

In certain embodiments, it is also possible that one or more (x)D(y),(x)D(y)T motifs and/or particular flanking sequences added to largermolecules will increase extravasation into tissues. In light of thepresent disclosure, peptides containing (x)D(y) and/or (x)D(y)Tsequences being tested as anti-inflammatory or anti-metastatic agents(Jackson et al., 1997; Maeda et al., 1997; Greenspoon et al., 1994)should be monitored for both increased extravasation and for toxiceffects on vasculature. However, in certain embodiments, it may bedesirable to produce proteinaceous compositions that enhanceextravasation into tissues. Improvement in extravasation of atherapeutic composition, or promoting extravasation for a therapeuticcomposition with a protein, polypeptide or peptide of the presentinvention may allow greater access of the therapeutic agent to tissues.Thus, methods of enhancing or decreasing extravasation of one or moreproteins, polypeptides, peptides or therapeutic agents are provided.Preferred therapeutic agents include, but are not limited to one or moreITs, antibodies, cytokines, virus, drugs and the like.

To produce peptides, polypeptides or proteins that lack the (x)D(y)and/or (x)D(y)T sequence, one could delete or mutate the conservedaspartic acid (D), substitute another amino acid for the aspartic acid,or insert one or more amino acids at or adjacent to its position. Anyamino acid that may replace the (D) residue in the sequence as aconsequence of a deletion or mutation event.

Alternatively the (x) residue could be deleted, substituted, or moved bythe insertion of one or more amino acids, to remove the (x)D(y) and/or(x)D(y)T sequence. Any amino acid that may replace the (x) residue inthe sequence as a consequence of the deletion or mutation event ispreferably not leucine (L), isoleucine (I), glycine (G) or valine (V).

Or the (y) residue could be deleted, substituted, or moved by theinsertion of one or more amino acids, to remove the (x)D(y) and/or(x)D(y)T sequence. Any amino acid that may replace the (y) residue inthe sequence as a consequence of the deletion or mutation event ispreferably not valine (V), leucine (L) or serine (S).

Additionally, the (x)D(y) and/or (x)D(y)T sequences can be removed byany mutation that alters or changes this sequence. Such mutationsinclude but are not limited to truncations, insertions, substitutionsand deletions of amino acids. It is contemplated that chemicalmodification may also alter a (x)D(y) and/or (x)D(y)T sequence to reduceits ability to induce or promote VLS.

Thus, it is contemplated that such mutations that affects the (x)D(y)sequence or flanking sequence may alter the ability of a polypeptide topromote VLS or other abilities associated with these sequences. Forexample, one preferred agent that produced VLS is abrin A chain (GenBankAccession number X76721; SEQ ID NO:3), which contains an IDV sequence atpositions 68-70 of its amino acid sequence. A glycine (G) is at position67. Therefore, a deletion of the isoleucine at position 68 would resultin the glycine at position 67 to be directly adjacent to the asparticacid residue (D) at original position 69. The new sequence created wouldthen be GDV at positions 67-69 of the mutated abrin A chain. This newtripeptide sequence still matches the VLS-inducing sequence (x)D(y)and/or (x)D(y)T. However, it is contemplated that since such a deletionwould shift the position of the tri-amino acid sequence in the structureof the mutated abrin A chain protein, polypeptide or peptide beingproduced. A shift in the position of the tri-amino acid sequence maymove it into a less favorable position to contact any cell, receptor ormolecule to promote or induce VLS. The resulting mutated abrin A chainprotein, polypeptide or peptide may have a reduced ability to promote orinduce VLS, and thus would be encompassed by the present invention.

Similarly, other toxins or compounds that induce VLS, including but notlimited to those listed in Table 1, can be mutated so that one or more(x)D(y) and/or one or more flanking residues are removed (i.e.,mutated). However, it is contemplated that to produce toxins orcompounds that have a reduced ability to induce VLS, it is preferablethat any remaining (x)D(y) and/or (x)D(y)T sequences to have a reducedexposure to the surface of the protein, polypeptide or peptide.

For example, it is contemplated that (x)D(y) and/or (x)D(y)T sequencesthat are at least partly located in the non-exposed portions of aprotein, polypeptide or peptide, or otherwise masked from full orpartial exposure to the surface of the molecule, would interact lesswith cells, receptors or other molecules to promote or induce VLS. Thus,it is contemplated that the complete elimination of (x)D(y) and/or(x)D(y)T sequences from the primary structure of the protein,polypeptide or peptide is not necessary to produce toxins or moleculeswith a reduced ability to induce or promote VLS. However, removal of all(x)D(y) and/or (x)D(y)T sequences is preferred to insure the compositionhas the least ability to induce or promote VLS.

To determine whether a mutation would likely produce a protein,polypeptide or peptide with a less exposed (x)D(y) and/or (x)D(y)Tmotif, the putative location of the moved or added (x)D(y) and/or(x)D(y)T sequence could be determined by comparison of the mutatedsequence to that of the unmutated protein, polypeptide or peptide'ssecondary and tertiary structure, as determined by such methods known tothose of ordinary skill in the art including, but not limited to, X-raycrystallography, NMR or computer modeling. Computer models of variouspolypeptide and peptide structures are also available in the literatureor computer databases. In a non-limiting example, the Entrez databasemay be used by one of ordinary skill in the art to identify targetsequences and regions for mutagenesis. The Entrez database iscrosslinked to a database of 3-D structures for the identified aminoacid sequence, if known. Such molecular models may be used to identify(x)D(y), (x)D(y)T and/or flanking sequences in peptides and polypeptidesthat are more exposed to contact with external molecules, (e.g.receptors) than similar sequences embedded in the interior of thepolypeptide or polypeptide. It is contemplated that (x)D(y), (x)D(y)Tand/or flanking sequences that are more exposed to contact with externalmolecules are more likely to contribute to promoting or reducing VLS andother toxic effects associated with these sequences, thus should beprimary targets for mutagenesis. In certain embodiments, when adding atleast one (x)D(y), (x)D(y)T and/or flanking sequence is desirable,regions of the protein that are more exposed to contact with externalmolecules are preferred as sites to add such a sequence. The mutated orwild-type protein, polypeptide or peptide's structure could bedetermined by X-ray crystallography or NMR directly before use in invitro or in vivo assays, as would be known to one of ordinary skill inthe art.

Once an amino acid sequence comprising a (x)D(y) and/or (x)D(y)Tsequence is altered in a peptide, polypeptide or protein, or added to apeptide, polypeptide or protein, changes in its ability to promote atleast one toxic effect may be assayed by any of the techniques describedherein or as would be known to one of ordinary skill in the art.

As used herein, “alter”, “altered”, “altering”, “alteration” of an aminoacid sequence comprising a (x)D(y) sequence or a (x)D(y)T sequence mayinclude chemical modification of an amino acid sequence comprising a(x)D(y) and/or a (x)D(y)T sequence in a protein, polypeptide or peptideas would be known to those of ordinary skill in the art, as well as anymutation of such an amino acid sequence including but not limited toinsertions, deletions, truncations, or substitutions. It is preferredthat such changes alters at least one toxic effect (i.e., the ability topromote VLS, EC damage, apoptosis, disintigrin-like activity) of one ormore amino acid sequence(s) comprising a (x)D(y) and/or (x)D(y)Tsequences. As used herein an amino acid sequence comprising a (x)D(y)sequence or a (x)D(y)T sequence may comprise at least one flankingsequence C- and/or N-terminal to a (x)D(y) and/or a (x)D(y)T tri- orquatra-peptide sequence. Such an “alteration” may be made in synthesizedpeptides, or in nucleic acid sequences that are expressed to producemutated proteins, polypeptides or peptides.

In an aspect of the invention, the alteration of an amino acid sequencecomprising a (x)D(y) and/or a (x)D(y)T sequence comprises removal of theamino acid sequence. As used herein “remove”, “removed”, “removing” or“removal” of an amino acid sequence comprising a (x)D(y) and/or a(x)D(y)T sequence refers to a mutation in the primary amino acidsequence that eliminates the presence of the (x)D(y) and/or a (x)D(y)Ttri- or quatra-peptide sequence, and/or at least one native flankingsequence. The terms “removed” or “lacks” may be used interchangably.

For example, it is contemplated that mutations including but not limitedto at least one insertion or substitution of at least one amino acidselected from the group phenylalanine (F); cysteine/cystine (C);methionine (M); alanine (A); threonine (T); serine (S); tryptophan (W);tyrosine (Y); proline (P); histidine (H); glutamic acid (E); glutamine(Q); aspartic acid (D); asparagine (N); lysine (K); and arginine (R),and including, but not limited to, those shown at Table 2 at theposition (x) of one or more (x)D(y) and/or (x)D(y)T sequences wouldreduce its ability to promote VLS. Table 2 below lists exemplary, butnot limiting, modified or unusual amino acids that are contemplated asuseful in certain aspects of the invention.

TABLE 2 Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. AminoAcid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine BAad 3-Aminoadipicacid Hyl Hydroxylysine BAla β-alanine, Ahyl Allo-Hydroxylysineβ-Amino-propionic acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid AileAllo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,sarcosine BAib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acidMeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelicacid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGlyN-Ethylglycine

It is also contemplated that mutations including but not limited to atleast one insertion or substitution of at least one amino acid selectedfrom the group isoleucine (I); valine (V); leucine (L); phenylalanine(F); cysteine/cystine (C); methionine (M); alanine (A); glycine (G);threonine (T); serine (S); tryptophan (W); tyrosine (Y); proline (P);histidine (H); glutamic acid (E); glutamine (Q); asparagine (N); lysine(K); and arginine (R), and including, but not limited to, those shown atTable 2 at the position (D) of one ore more (x)D(y) and/or (x)D(y)Tsequences would reduce its ability to promote VLS.

It is contemplated that mutations including but not limited to at leastone insertion or substitution of at least one amino acid selected fromthe group isoleucine (I); phenylalanine (F); cysteine/cystine (C);methionine (M); alanine (A); glycine (G); threonine (T); tryptophan (W);tyrosine (Y); proline (P); histidine (H); glutamic acid (E); glutamine(Q); aspartic acid (D); asparagine (N); lysine (K); and arginine (R),and including, but not limited to, those shown at Table 2 at theposition (y) of one or more (x)D(y) and/or (x)D(y)T sequences wouldreduce its ability to promote VLS.

Amino acids that flank either the (x) or (y) residue of the (x)D(y)sequence may also contribute to its ability to promote VLS. For example,is it contemplated that mutations including but not limited to at leastone insertion or substitution of at least one amino acid selected fromthe group isoleucine (I); valine (V); leucine (L); phenylalanine (F);cysteine/cystine (C); methionine (M); alanine (A); glycine (G); serine(S); tryptophan (W); tyrosine (Y); proline (P); histidine (H); glutamicacid (E); glutamine (Q); aspartic acid (D); asparagine (N); lysine (K);and arginine (R), and including, but not limited to, those shown atTable 2 at the position T of one or more (x)D(y)T sequences would reduceits ability to promote VLS.

It is further contemplated that at least one mutation, chemicalmodification, movement or other alteration in the N- or C-terminalflanking sequences of the (x)D(y) and/or (x)D(y)T sequence would alsoproduce proteins, polypeptides or peptides that have a reduced abilityto promote VLS. Preferably, such mutations or alterations would occur inone or more residues which will not effect the active site. In otherembodiments, the mutations or alterations would occur in one or moreresidues of from about 1, about 2, about 3, about 4, about 5, about 6 ormore N-terminal and/or C-terminal to the (x)D(y) tripeptide sequence. Inother aspects, one or more residues that are not adjacent to the (x)D(y)tripeptide may contribute to the function of the (x)D(y) motif. Suchresidues may be identified by their proximity to the tripeptide sequencein a 3-dimentional model, as described herein and as would be known toone of ordinary skill in the art, and are contemplated for alteration aspart of a flanking sequence. Such alterations may include any of thosedescribed above for altering the (x)D(y) and (x)D(y)T sequences, as longas one or more “wild type” flanking residues are altered, removed,moved, chemically modified, etc.

Proteins, polypeptides and peptides produced using the methods of thepresent invention that have a reduced ability to induce VLS would haveapplication in serving as protective agents against VLS produced bycompositions containing the (x)D(y) and/or (x)D(y)T sequence. It iscontemplated that such proteins, polypeptides and peptides may serve asinhibitors that block the activity of the (x)D(y) and/or (x)D(y)Tsequence. Additionally, such proteins, polypeptides and peptides may beused in the creation of ITs with a reduced ability to produce VLS.

1. Mutagenesis

In certain aspects, mutagenesis of nucleic acids encoding peptides,polypeptides or proteins may be used to produce the desired mutations toenhance or reduce a composition's ability to promote VLS, apoptosis orother effects associated with the (x)D(y) and flanking sequences.Mutagenesis may be conducted by any means disclosed herein or known toone of ordinary skill in the art.

One particularly useful mutagenesis technique is alanine scanningmutagenesis in which a number of residues are substituted individuallywith the amino acid alanine so that the effects of losing side-chaininteractions can be determined, while minimizing the risk of large-scaleperturbations in protein conformation (Cunningham et al., 1989).

As specific amino acids may be targeted, site-specific mutagenesis is atechnique useful in the preparation of individual peptides, orbiologically functional equivalent proteins or peptides, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of themutation site being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art. As will be appreciated, the technique typically employs abacteriophage vector that exists in both a single stranded and doublestranded form. Typical vectors useful in site-directed mutagenesisinclude vectors such as the M13 phage. These phage vectors arecommercially available and their use is generally well known to thoseskilled in the art. Double stranded plasmids are also routinely employedin site directed mutagenesis, which eliminates the step of transferringthe gene of interest from a phage to a plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, and subjected to DNA polymerizingenzymes such as E. coli polymerase I Klenow fragment, in order tocomplete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement. Alternatively, a pairof primers may be annealed to two separate strands of a double strandedvector to simultaneously synthesize both corresponding complementarystrands with the desired mutation(s) in a PCR™ reaction.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

2. Recombinant Vectors, Host Cells and Expression

The term “expression vector or construct” means any type of geneticconstruct containing a nucleic acid coding for a gene product in whichpart or all of the nucleic acid encoding sequence is capable of beingtranscribed. The transcript may be translated into a protein, but itneed not be. Thus, in certain embodiments, expression includes bothtranscription of a gene and translation of a RNA into a gene product. Inother embodiments, expression only includes transcription of the nucleicacid, for example, to generate antisense constructs.

Particularly useful vectors are contemplated to be those vectors inwhich the coding portion of the DNA segment, whether encoding a fulllength protein, polypeptide or smaller peptide, is positioned under thetranscriptional control of a promoter. A “promoter” refers to a DNAsequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrases “operatively positioned”, “undercontrol” or “under transcriptional control” means that the promoter isin the correct location and orientation in relation to the nucleic acidcoding for the gene product to control RNA polymerase initiation andexpression of the gene.

The promoter may be in the form of the promoter that is naturallyassociated with a gene, as may be obtained by isolating the 5′non-coding sequences located upstream of the coding segment or exon, forexample, using recombinant cloning and/or PCR™ technology, in connectionwith the compositions disclosed herein (PCR™ technology is disclosed inU.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, each incorporatedherein by reference).

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a gene in its natural environment. Suchpromoters may include promoters normally associated with other genes,and/or promoters isolated from any other bacterial, viral, eukaryotic,or mammalian cell, and/or promoters made by the hand of man that are not“naturally occurring,” i.e., containing difference elements fromdifferent promoters, or mutations that increase, decrease, or alterexpression.

Naturally, it will be important to employ a promoter that effectivelydirects the expression of the DNA segment in the cell type, organism, oreven animal, chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology, for example, see Sambrook et al.,(1989), incorporated herein by reference. The promoters employed may beconstitutive, or inducible, and can be used under the appropriateconditions to direct high level expression of the introduced DNAsegment, such as is advantageous in the large-scale production ofrecombinant proteins or peptides.

At least one module in a promoter generally functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as the promoter forthe mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes, a discrete element overlying the startsite itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have beenshown to contain functional elements downstream of the start site aswell. The spacing between promoter elements frequently is flexible, sothat promoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter that is employed to control the expression of anucleic acid is not believed to be critical, so long as it is capable ofexpressing the nucleic acid in the targeted cell. Thus, where a humancell is targeted, it is preferable to position the nucleic acid codingregion adjacent to and under the control of a promoter that is capableof being expressed in a human cell. Generally speaking, such a promotermight include either a human or viral promoter.

In expression, one will typically include a polyadenylation signal toeffect proper polyadenylation of the transcript. The nature of thepolyadenylation signal is not believed to be crucial to the successfulpractice of the invention, and any such sequence may be employed.Preferred embodiments include the SV40 polyadenylation signal and thebovine growth hormone polyadenylation signal, convenient and known tofunction well in various target cells. Also contemplated as an elementof the expression cassette is a terminator. These elements can serve toenhance message levels and to minimize read through from the cassetteinto other sequences.

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

It is contemplated that proteins, polypeptides or peptides may beco-expressed with other selected proteins, wherein the proteins may beco-expressed in the same cell or a gene(s) may be provided to a cellthat already has another selected protein. Co-expression may be achievedby co-transfecting the cell with two distinct recombinant vectors, eachbearing a copy of either of the respective DNA. Alternatively, a singlerecombinant vector may be constructed to include the coding regions forboth of the proteins, which could then be expressed in cells transfectedwith the single vector. In either event, the term “co-expression” hereinrefers to the expression of both the gene(s) and the other selectedprotein in the same recombinant cell.

As used herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous DNAsegment or gene, such as a cDNA or gene encoding a protein has beenintroduced. Therefore, engineered cells are distinguishable fromnaturally occurring cells which do not contain a recombinantlyintroduced exogenous DNA segment or gene. Engineered cells are thuscells having a gene or genes introduced through the hand of man.Recombinant cells include those having an introduced cDNA or genomicgene, and also include genes positioned adjacent to a promoter notnaturally associated with the particular introduced gene.

To express a recombinant protein, polypeptide or peptide, whether mutantor wild-type, in accordance with the present invention one would preparean expression vector that comprises a wild-type, or mutantprotein-encoding nucleic acid under the control of one or morepromoters. To bring a coding sequence “under the control of” a promoter,one positions the 5′ end of the transcription initiation site of thetranscriptional reading frame generally between about 1 and about 50nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. The“upstream” promoter stimulates transcription of the DNA and promotesexpression of the encoded recombinant protein. This is the meaning of“recombinant expression” in this context.

Many standard techniques are available to construct expression vectorscontaining the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieveprotein, polypeptide or peptide expression in a variety ofhost-expression systems. Cell types available for expression include,but are not limited to, bacteria, such as E. coli and B. subtilistransformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors.

Certain examples of prokaryotic hosts are E. coli strain RR1, E. coliLE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coliW3110 (F-, lambda-, prototrophic, ATCC No. 273325); bacilli such asBacillus subtilis; and other enterobacteriaceae such as Salmonellatyphimurium, Serratia marcescens, and various Pseudomonas species.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli is oftentransformed using derivatives of pBR322, a plasmid derived from an E.coli species. pBR322 contains genes for ampicillin and tetracyclineresistance and thus provides easy means for identifying transformedcells. The pBR plasmid, or other microbial plasmid or phage must alsocontain, or be modified to contain, promoters which can be used by themicrobial organism for expression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEM™-11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as E. coli LE392.

Further useful vectors include pIN vectors (Inouye et al., 1985); andpGEX vectors, for use in generating glutathione S-transferase (GST)soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with β-galactosidase,ubiquitin, and the like.

Promoters that are most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase), lactose and tryptophan (trp)promoter systems. While these are the most commonly used, othermicrobial promoters have been discovered and utilized, and detailsconcerning their nucleotide sequences have been published, enablingthose of skill in the art to ligate them functionally with plasmidvectors.

The following details concerning recombinant protein production inbacterial cells, such as E. coli, are provided by way of exemplaryinformation on recombinant protein production in general, the adaptationof which to a particular recombinant expression system will be known tothose of skill in the art.

Bacterial cells, for example, E. coli, containing the expression vectorare grown in any of a number of suitable media, for example, LB. Theexpression of the recombinant protein may be induced, e.g., by addingIPTG to the media or by switching incubation to a higher temperature.After culturing the bacteria for a further period, generally of between2 and 24 h, the cells are collected by centrifugation and washed toremove residual media.

The bacterial cells are then lysed, for example, by disruption in a cellhomogenizer and centrifuged to separate the dense inclusion bodies andcell membranes from the soluble cell components. This centrifugation canbe performed under conditions whereby the dense inclusion bodies areselectively enriched by incorporation of sugars, such as sucrose, intothe buffer and centrifugation at a selective speed.

If the recombinant protein is expressed in the inclusion bodies, as isthe case in many instances, these can be washed in any of severalsolutions to remove some of the contaminating host proteins, thensolubilized in solutions containing high concentrations of urea (e.g. 8M) or chaotropic agents such as guanidine hydrochloride in the presenceof reducing agents, such as β-mercaptoethanol or DTT (dithiothreitol).

It is contemplated that the proteins, polypeptides or peptides producedby the methods of the invention may be “overexpressed”, i.e., expressedin increased levels relative to its natural expression in cells. Suchoverexpression may be assessed by a variety of methods, includingradio-labeling and/or protein purification. However, simple and directmethods are preferred, for example, those involving SDS/PAGE and proteinstaining or western blotting, followed by quantitative analyses, such asdensitometric scanning of the resultant gel or blot. A specific increasein the level of the recombinant protein, polypeptide or peptide incomparison to the level in natural cells is indicative ofoverexpression, as is a relative abundance of the specific protein,polypeptides or peptides in relation to the other proteins produced bythe host cell and, e.g., visible on a gel.

3. Proteins, Polypeptides, and Peptides

The present invention also provides purified, and in preferredembodiments, substantially purified, proteins, polypeptides, orpeptides. The term “purified proteins, polypeptides, or peptides” asused herein, is intended to refer to an proteinaceous composition,isolatable from mammalian cells or recombinant host cells, wherein theat least one protein, polypeptide, or peptide is purified to any degreerelative to its naturally-obtainable state, i.e., relative to its puritywithin a cellular extract. A purified protein, polypeptide, or peptidetherefore also refers to a wild-type or mutant protein, polypeptide, orpeptide free from the environment in which it naturally occurs.

The nucleotide and protein, polypeptide and peptide sequences forvarious genes have been previously disclosed, and may be found atcomputerized databases known to those of ordinary skill in the art. Onesuch database is the National Center for Biotechnology Information'sGenbank and GenPept databases. The coding regions for these known genesmay be amplified and/or expressed using the techniques disclosed hereinor by any technique that would be know to those of ordinary skill in theart. Additionally, peptide sequences may be synthesized by methods knownto those of ordinary skill in the art, such as peptide synthesis usingautomated peptide synthesis machines, such as those available fromApplied Biosystems (Foster City, Calif.).

Generally, “purified” will refer to a specific protein, polypeptide, orpeptide composition that has been subjected to fractionation to removevarious other proteins, polypeptides, or peptides, and which compositionsubstantially retains its activity, as may be assessed, for example, bythe protein assays, as described herein below, or as would be known toone of ordinary skill in the art for the desired protein, polypeptide orpeptide.

Where the term “substantially purified” is used, this will refer to acomposition in which the specific protein, polypeptide, or peptide formsthe major component of the composition, such as constituting about 50%of the proteins in the composition or more. In preferred embodiments, asubstantially purified protein will constitute more than 60%, 70%, 80%,90%, 95%, 99% or even more of the proteins in the composition.

A peptide, polypeptide or protein that is “purified to homogeneity,” asapplied to the present invention, means that the peptide, polypeptide orprotein has a level of purity where the peptide, polypeptide or proteinis substantially free from other proteins and biological components. Forexample, a purified peptide, polypeptide or protein will often besufficiently free of other protein components so that degradativesequencing may be performed successfully.

Various methods for quantifying the degree of purification of proteins,polypeptides, or peptides will be known to those of skill in the art inlight of the present disclosure. These include, for example, determiningthe specific protein activity of a fraction, or assessing the number ofpolypeptides within a fraction by gel electrophoresis.

To purify a desired protein, polypeptide, or peptide a natural orrecombinant composition comprising at least some specific proteins,polypeptides, or peptides will be subjected to fractionation to removevarious other components from the composition. In addition to thosetechniques described in detail herein below, various other techniquessuitable for use in protein purification will be well known to those ofskill in the art. These include, for example, precipitation withammonium sulfate, PEG, antibodies and the like or by heat denaturation,followed by centrifugation; chromatography steps such as ion exchange,gel filtration, reverse phase, hydroxylapatite, lectin affinity andother affinity chromatography steps; isoelectric focusing; gelelectrophoresis; and combinations of such and other techniques.

Another example is the purification of a specific fusion protein using aspecific binding partner. Such purification methods are routine in theart. As the present invention provides DNA sequences for the specificproteins, any fusion protein purification method can now be practiced.This is exemplified by the generation of an specific protein-glutathioneS-transferase fusion protein, expression in E. coli, and isolation tohomogeneity using affinity chromatography on glutathione-agarose or thegeneration of a polyhistidine tag on the N- or C-terminus of theprotein, and subsequent purification using Ni-affinity chromatography.However, given many DNA and proteins are known, or may be identified andamplified using the methods described herein, any purification methodcan now be employed.

Although preferred for use in certain embodiments, there is no generalrequirement that the protein, polypeptide, or peptide always be providedin their most purified state. Indeed, it is contemplated that lesssubstantially purified protein, polypeptide or peptide, which arenonetheless enriched in the desired protein compositions, relative tothe natural state, will have utility in certain embodiments.

Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein. Inactive products also have utility incertain embodiments, such as, e.g., in determining antigenicity viaantibody generation.

4. Antibodies

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally,IgG and/or IgM are preferred because they are the most common antibodiesin the physiological situation and because they are most easily made ina laboratory setting.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production, and their use isgenerally preferred. The invention thus provides monoclonal antibodiesof the human, murine, monkey, rat, hamster, rabbit and even chickenorigin. Due to the ease of preparation and ready availability ofreagents, murine monoclonal antibodies will often be preferred.

However, “humanized” antibodies are also contemplated, as are chimericantibodies from mouse, rat, or other species, bearing human constantand/or variable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof. Methods for the developmentof antibodies that are “custom-tailored” to the patient's disease arelikewise known and such custom-tailored antibodies are alsocontemplated.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic protein composition or comprising a target epitope inaccordance with the present invention and collecting antisera from thatimmunized animal.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include all acceptable immunostimulatory compounds, such ascytokines, toxins or synthetic compositions.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion is also contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, Pa.);low-dose Cyclophosphamide (CYP; 300 mg/m²) (Johnson/Mead, N.J.),cytokines such as γ-interferon, IL-2, or IL-12 or genes encodingproteins involved in immune helper functions, such as B-7.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified protein, polypeptide, peptide or domain, be it awild-type or mutant composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are preferred animals, however, the use ofrabbit, sheep or frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986, pp. 60-61), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

The animals are injected with antigen, generally as described above. Theantigen may be coupled to carrier molecules such as keyhole limpethemocyanin if necessary. The antigen would typically be mixed withadjuvant, such as Freund's complete or incomplete adjuvant. Boosterinjections with the same antigen would occur at approximately two-weekintervals.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible.

Often, a panel of animals will have been immunized and the spleen of ananimal with the highest antibody titer will be removed and the spleenlymphocytes obtained by homogenizing the spleen with a syringe.Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp2/0-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. First, a sample of the hybridomacan be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion (e.g., a syngeneic mouse).Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to provideMAbs in high concentration. Second, the individual cell lines could becultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiesof the invention can be obtained from the monoclonal antibodies soproduced by methods which include digestion with enzymes, such as pepsinor papain, and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It is also contemplated that a molecular cloning approach may be used togenerate monoclonals. For this, combinatorial immunoglobulin phagemidlibraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and controlcells. The advantages of this approach over conventional hybridomatechniques are that approximately 10⁴ times as many antibodies can beproduced and screened in a single round, and that new specificities aregenerated by H and L chain combination which further increases thechance of finding appropriate antibodies.

Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer, orby expression of full-length gene or of gene fragments in E. coli.

C. ITs

Toxins and/or Mabs may be derived from natural sources or produced usingrecombinant DNA technology. At least one toxin and at least one antibodymay be combined to form an “ITn”. ITs combine into a single molecule,the exquisite specificity of a ligand and the extraordinary toxicity ofa toxin. Despite their conceptual simplicity, ITs are large and complexmolecules that are continually undergoing improvements for optimal invivo activity since each of their common components, e.g., one or more abinding moieties, one or more cross-linkers, and one or more toxins,introduces a different set of problems that must be addressed for the ITto function optimally in vivo.

Any antibody of sufficient selectivity, specificity or affinity may beemployed as the basis for an IT. Such properties may be evaluated usingconventional immunological screening methodology known to those of skillin the art. Sites for binding to biological active molecules in theantibody molecule, in addition to the canonical antigen binding sites,include sites that reside in the variable domain that can bindpathogens, B-cell superantigens, the T cell co-receptor CD4 and theHIV-1 envelope (Sasso el al., 1989; Shorki et al., 1991; Silvermann etal., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al.,1993; Kreier et al., 1991). In addition, the variable domain is involvedin antibody self-binding (Kang et al., 1988), and contains epitopes(idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

The origin or derivation of the antibody or antibody fragment for use inthe invention (e.g., Fab′, Fab or F(ab′)₂) is not crucial to thepractice of the invention, so long as the antibody or fragment that isemployed has the desired properties for the ultimately intended use ofthe IT. Thus, where monoclonal antibodies are employed, they may be ofhuman, murine, monkey, rat, hamster, chicken or even rabbit origin. Theinvention also contemplates the use of human antibodies, “humanized” orchimeric antibodies from mouse, rat, or other species, bearing humanconstant and/or variable region domains, single chain antibodies, Fvdomains, as well as recombinant antibodies and fragments thereof. Ofcourse, due to the ease of preparation and ready availability ofreagents, murine Mabs will typically be preferred.

In certain therapeutic embodiments, one may use known antibodies, suchas those having high selectivity for solid tumors, such as B72.3, PRBC5or PR4D2 for colorectal tumors; HMFG-2, TAG 72, SM-3, or anti-p185.sup.Her2 for breast tumors; anti-p 185.sup.Her2 for lung tumors;9.2.27 for melanomas; MO v18 and OV-TL3 for ovarian tumors, and anti-Id,CD19, CD22, CD25, CD7 and CD5 for lymphomas and leukemias. Anti-CD2,anti-CD25, anti-CD4 and anti-CD45R° ITs may be purified according to theinvention and used to kill malignant T cells or HIV-infected cells.Also, CD3-specific ITs as well as CD4 and CD25 specific ITs may bepurified and used to prevent acute GVHD after bone marrowtransplantation.

In other embodiments, one may use another immunogen and prepare a newMab. The technique for preparing Mabs is quite straightforward, and maybe readily carried out using techniques well known to those of skill inthe art, as exemplified by the technique of Kohler & Milstein (1975).Generally, immunogens are injected intraperitoneally into mice. Thisprocess is repeated three times at two-weekly intervals, the finalimmunization being by the intravenous route. Three days later the spleencells are harvested and fused with SP2/0 myeloma cells by standardprotocols (Kohler & Milstein, 1975): Hybridomas producing antibodieswith the appropriate reactivity are then cloned by limiting dilution.

The toxins that have been used to form ITs are derived from bacteria orplants and are inhibitors of protein synthesis. They are among the mostpowerful cell poisons known. Fewer than ten molecules will kill a cellif they enter the cytosol (although many times that number must bind tothe cell surface because the entry process is inefficient). Thisextraordinary potency initially led to the concern that such poisonswere too powerful to control. However, the toxins can be renderedinnocuous (except when directed to the target cells) simply by removingor modifying their cell-binding domain or subunit. The remaining portionof the toxin (lacking a cell-binding domain) is then coupled to a ligand(e.g., an antibody) that targets the toxic portion to the target cell.By selecting an antibody lacking unwanted cross-reactivity, ITs aresafer and have fewer non-specific cytotoxic effects than mostconventional anticancer drugs. The other main attraction of toxins isthat because they are inhibitors of protein synthesis, they kill restingcells as efficiently as dividing cells. Hence, tumor or infected cellsthat are not in cycle at the time of treatment do not escape thecytotoxic effect of an IT.

“Toxin” is employed herein to mean any anticellular agent, and includesbut is not limited to cytotoxins and any combination of anticellularagents. In the case of chemotherapeutic agents, agents such as ahormone, a steroid for example; an antimetabolite such as cytosinearabinoside, fluorouracil, methotrexate or aminopterin; ananthracycline; mitomycin C; a vinca alkaloid; demecolcine; etoposide;mithramycin; or an antitumor alkylating agent such as chlorambucil ormelphalan, may be used.

However, preferred toxins will be plant-, fungus- or bacteria-derivedtoxins, which, by way of example, include various A chain toxins,particularly RTA; RIPs such as saporin or gelonin, α-sarcin, aspergillinor restrictocin; ribonucleases such as placental ribonuclease;angiogenin, diphtheria toxin, and Pseudomonas exotoxin, to name just afew. The exemplary toxins that can be mutated to remove or alter theplacement of sequences that induce VLS are listed in Table 1.

Plant holotoxin often contain two disulfide-bonded chains, the A and Bchains. The B chain carries both a cell-binding region (whose receptoris often uncharacterized) and a translocation region, which facilitatesthe insertion of the A chain through the membrane of an acidintracellular compartment into the cytosol. The A chain then kills thecell after incorporation. For their use in vivo, the ligand and toxinmust be coupled in such a way as to remain stable while passing throughthe bloodstream and the tissues and yet be labile within the target cellso that the toxic portion can be released into the cytosol.

The most preferred toxin moiety for use in connection with the inventionis RTA, and particularly toxin A chain which has been treated to modifyor remove carbohydrate residues, so-called dgRTA. Recombinant A chainexpressed in E. coli and also lacking carbohydrates can be used. Incertain embodiments, RTA may be made as described herein below inExample 3.

However, it may be desirable from a pharmacologic standpoint to employthe smallest molecule possible that nevertheless provides an appropriatebiological response. One may thus desire to employ smaller A chainpeptides or other toxins which will provide an adequate anti-cellularresponse. To this end, it has been discovered by others that RTA may be“truncated” by the removal of 30 N-terminal amino acids by Nagarase(Sigma), and still retain an adequate toxin activity. It is proposedthat where desired, this truncated A chain may be employed in conjugatesin accordance with the invention.

Alternatively, one may find that the application of recombinant DNAtechnology to the toxin moiety will provide additional significantbenefits in accordance the invention. In that the cloning and expressionof biologically active RTA and other VLS-inducing toxins have now beenenabled through the publications of others (O'Hare et al., 1987; Lamb etal., 1985; Halling et al., 1985), it is now possible to identify andprepare smaller or otherwise variant peptides which nevertheless exhibitan appropriate toxin activity. Moreover, the fact that RTA and otherVLS-inducing toxins have now been cloned allows the application ofsite-directed mutagenesis, through which one can readily prepare andscreen for A chain and other VLS-inducing toxins, toxin-derived peptidesand obtain additional useful moieties for use in connection with thepresent invention. Once identified, these moieties can be mutated toproduce toxins with a reduced ability to promote VLS, apoptosis,disintegrin-like activity, EC damaging activity and other effects ofsuch sequences described herein or known to one of skill in the art.

Fusion-ITs with PE, DT-A, etc. in any combination are made byrecombinant DNA technology as would be known to one of ordinary skill inthe art. Antibodies, cytokines or soluble receptor DNA may be used insuch preparations.

The cross-linking of many, but not all toxins, of the conjugate with thebinding agent region is an important aspect of the invention. In thecase of RTA, if one desires a conjugate having biological activity, itis believed that a cross-linker which presents a disulfide function isrequired. The reason for this is unclear, but is likely due to a needfor the toxin moiety to be readily releasable from the binding agentonce the agent has “delivered” the toxin inside the targeted cells. Eachtype of cross-linker, as well as how the cross-linking is performed,will tend to vary the pharmacodynamics of the resultant conjugate.Ultimately, one desires to have a conjugate that will remain intactunder conditions found everywhere in the body except the intended siteof action, at which point it is desirable that the conjugate have good“release” characteristics. Therefore, the particular cross-linkingscheme, including in particular the particular cross-linking reagentused and the structures that are cross-linked, will be of somesignificance.

Cross-linking reagents are used to form molecular bridges that tietogether functional groups of two different proteins (e.g., a toxin anda binding agent). To link two different proteins in a step-wise manner,heterobifunctional cross-linkers can be used which eliminate theunwanted homopolymer formation. An exemplary heterobifunctionalcross-linker contains two reactive groups: one reacting with primaryamine group (e.g., N-hydroxy succinimide) and the other reacting with athiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.).Through the primary amine reactive group, the cross-linker may reactwith the lysine residue(s) of one protein (e.g., the selected antibodyor fragment) and through the thiol reactive group, the crosslinker,already tied up to the first protein, reacts with the cysteine residue(free sulfhydryl group) of the other protein (e.g., dgRTA).

The spacer arm between these two reactive groups of any cross-linkersmay have various length and chemical composition. A longer spacer armallows a better flexibility of the conjugate components while someparticular components in the bridge (e.g., benzene group) may lend extrastability to the reactive group or an increased resistance of thechemical link to the action of various aspects (e.g., disulfide bondresistant to reducing agents).

The most preferred cross-linking reagent is SMPT, which is abifunctional cross-linker containing a disulfide bond that is“sterically hindered” by an adjacent benzene ring and methyl groups. Itis believed that steric hindrance of the disulfide bond serves afunction of protecting the bond from attack by thiolate anions such asglutathione which can be present in tissues and blood, and thereby helpin preventing decoupling of the conjugate prior to its delivery to thesite of action by the binding agent. The SMPT cross-linking reagent, aswith many other known cross-linking reagents, lends the ability tocrosslink functional groups such as the SH of cysteine or primary amines(e.g., the epsilon amino group of lysine). Another possible type ofcross-linker includes the heterobifunctional photoreactive phenylazidescontaining a cleavable disulfide bond such assulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3′-dithiopropionate.The N-hydroxy-succinimidyl group reacts with primary amino groups andthe phenylazide (upon photolysis) reacts non-selectively with any aminoacid residue.

Although the “hindered” cross-linkers will generally be preferred in thepractice of the invention, non-hindered linkers can be employed andadvantages in accordance herewith nevertheless realized. Other usefulcross-linkers, not considered to contain or generate a protecteddisulfide, include SATA, SPDP and 2-iminothiolane. The use of suchcross-linkers is well understood in the art.

1. IT Conjugates

The present invention provides ITs against target epitopes, such asepitopes expressed on a diseased tissue or a disease causing cell. Incertain embodiments the IT comprises at least one toxin describedherein. In other embodiments the IT or toxin further comprises at leasta second agent. Such an agent may be a molecule or moiety. Such amolecule or moiety may comprise, but is not limited to, at least oneeffector or reporter molecule. Effector molecules comprise moleculeshaving a desired activity, e.g., cytotoxic activity. Non-limitingexamples of effector molecules which have been attached to antibodiesinclude toxins, anti-tumor agents, therapeutic enzymes, radio-labelednucleotides, antiviral agents, chelating agents, cytokines, growthfactors, and oligo- or poly-nucleotides. By contrast, a reportermolecule is defined as any moiety which may be detected using an assay.Non-limiting examples of reporter molecules which have been conjugatedto antibodies include enzymes, radiolabels, haptens, fluorescent labels,phosphorescent molecules, chemiluminescent molecules, chromophores,luminescent molecules, photoaffinity molecules, colored particles orligands, such as biotin.

Certain examples of at least a second agent comprises at least onedetectable label. “Detectable labels” are compounds and/or elements thatcan be detected due to their specific functional properties, and/orchemical characteristics, the use of which allows the antibody to whichthey are attached to be detected, and/or further quantified if desired.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236;4,938,948; and 4,472,509, each incorporated herein by reference). Theimaging moieties used can be paramagnetic ions; radioactive isotopes;fluorochromes; NMR-detectable substances; X-ray imaging.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter& Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

2. Methods for IT Preparation

Methods of making and preparing ITs are known to those of ordinary skillin the art. Such methods are disclosed in U.S. Pat. No. 5,686,072, U.S.Pat. No. 5,578,706, U.S. Pat. No. 4,792,447, U.S. Pat. No. 5,045,451,U.S. Pat. No. 4,664,911, and U.S. Pat. No. 5,767,072, each incorporatedherein by reference). The toxin moiety of the IT may be any one of avariety of toxins that are commonly employed in the art. It may be anintact toxin, a toxin A chain, or a naturally occurring single-chainRIP. Toxins which are encompassed by the invention include, but are notlimited to, diphtheria toxin (DT) and DT(CRM-45); pseudomonas endotoxinderived PE38; RTA and abrin and blocked forms of both of these; geloninand saporin.

ITs comprising Mabs covalently bound to dgRTA by hindered disulfidelinkers have recently entered clinical trials for the treatment ofnon-Hodgkin's (B cell) lymphoma, Hodgkin's lymphoma neplasms or GVHD.These “second generation” ITs are stable, long lived and display potentcytotoxicity to target cells. Standardized procedures for rapidpreparation of high yields of these ITs have been developed (Ghetie etal., 1991).

The procedure for preparation of the ITs with, for example, dgRTAcomprises the derivitization of Mabs with SMPT and reduction of dgRTAwith dithiothreitol (DTT), followed by the reaction of the twocomponents to establish a hindered interchain disulfide bond. Thechemical crosslinking reaction results in a mixture of antibody, toxinand ITs which are then purified, initially to remove the free antibodyand free toxin molecules and subsequently to separate the different ITspecies which comprise one antibody molecule conjugated with one, two,three or more than three toxin molecules, respectively. The unreactedcomponents of the crosslinking reaction may be removed by successivechromatographies on an affinity chromatography column such as activateddye/agarose to remove free antibody followed by gel filtration to removehigh molecular weight material and free toxin.

The result of this procedure is a mixture of conjugates of varioustoxin/antibody ratios. An important embodiment of the present inventionis the further purification of this mixture to obtain preparationsessentially comprising ITs of a single toxin/antibody ratio separatedfrom ITs of different toxin/antibody ratios. This purification isaccomplished by further chromatographic separation which may beaccomplished by affinity chromatography for example, using a saltgradient to elute the various species of ITs and gel filtration toseparate the ITs from larger molecules.

Another important embodiment of the present invention is the ability todetermine which of the toxin/IgG ratios is the most effective cytotoxicagent to be used in pharmacological preparations. The isolation andcharacterization of each of the single species of IT made possible bythe present invention is of particular advantage in clinicalapplications as it allows the practitioner to exercise more precisecontrol over the effective amount of IT to be administered in aparticular situation.

3. Gel Filtration

A gel to be used in the procedures of the present invention is a threedimensional network which has a random structure. Molecular sieve gelscomprise cross-linked polymers that do not bind or react with thematerial being analyzed or separated. For gel filtration purposes, thegel material is generally uncharged. The space within the gel is filledwith liquid and the liquid phase constitutes the majority of the gelvolume. Materials commonly used in gel filtration columns includedextran, agarose and polyacrylamide.

Dextran is a polysaccharide composed of glucose residues and iscommercially available under the name SEPHADEX (Phamacia Fine Chemicals,Inc.). The beads are prepared with various degrees of cross-linking inorder to separate different sized molecules by providing various poresizes. Alkyl dextran is cross-linked with N,N′-methylenebisacrylamide toform SEPHACRYL-S100 to S1000 which allows strong beads to be made thatfractionate in larger ranges than SEPHADEX can achieve.

Polyacrylamide may also be used as a gel filtration medium.Polyacrylamide is a polymer of cross-linked acrylamide prepared withN,N′-methylenebisacrylamide as the cross-linking agent. Polyacrylamideis available in a variety of pore sizes from Bio-Rad Laboratories (USA)to be used for separation of different size particles.

The gel material swells in water and in a few organic solvents. Swellingis the process by which the pores become filled with liquid to be usedas eluant. As the smaller molecules enter the pores, their progressthrough the gel is retarded relative to the larger molecules which donot enter the pores. This is the basis of the separation. The beads areavailable in various degrees of fineness to be used in differentapplications. The coarser the bead, the faster the flow and the poorerthe resolution. Superfine is to be used for maximum resolution, but theflow is very slow. Fine is used for preparative work in large columnswhich require a faster flow rate. The coarser grades are for largepreparations in which resolution is less important than time, or forseparation of molecules with a large difference in molecular weights.For a discussion of gel chromatography, see Freifelder, PhysicalBiochemistry, Second Edition, pages 238-246, incorporated herein byreference.

The most preferred methods of gel filtration for use in the presentinvention are those using dextran gels, such as SEPHADEX, and thoseusing dextran-polyacrylamide gels such as SEPHACRYL which are able toseparate molecules in the 180 to 240 kilodalton range.

4. Affinity Chromatography

Affinity chromatography is generally based on the recognition of aprotein by a substance such as a ligand or an antibody. The columnmaterial may be synthesized by covalently coupling a binding molecule,such as an activated dye, for example to an insoluble matrix. The columnmaterial is then allowed to adsorb the desired substance from solution.Next, the conditions are changed to those under which binding does notoccur and the substrate is eluted. The requirements for successfulaffinity chromatography are that the matrix must adsorb molecules, theligand must be coupled without altering its binding activity, a ligandmust be chosen whose binding is sufficiently tight, and it must bepossible to elute the substance without destroying it.

A preferred embodiment of the present invention is an affinitychromatography method wherein the matrix is a reactive dye-agarosematrix. Blue-SEPHAROSE, a column matrix composed of Cibacron Blue 3GAand agarose or SEPHAROSE may be used as the affinity chromatographymatrix. The most preferred matrix is SEPHAROSE CL-6B available asReactive Blue 2 from Sigma Chemical Company, catalogue #R 8752. Thismatrix binds the ITs of the present invention directly and allows theirseparation by elution with a salt gradient.

5. ELISA

ELISAs may be used in conjunction with the invention. In an ELISA assay,proteins or peptides incorporating toxin A chain sequences areimmobilized onto a selected surface, preferably a surface exhibiting aprotein affinity such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed material, it is desirableto bind or coat the assay plate wells with a nonspecific protein that isknown to be antigenically neutral with regard to the test antisera suchas bovine serum albumin (BSA), casein or solutions of milk powder. Thisallows for blocking of nonspecific adsorption sites on the immobilizingsurface and thus reduces the background caused by nonspecific binding ofantisera onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/TWEEN. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from 2 to 4 hours, at temperatures preferably on the order of 25° C.to 37° C. Following incubation, the antisera-contacted surface is washedso as to remove non-immunocomplexed material. A preferred washingprocedure includes washing with a solution such as PBS/TWEEN, or boratebuffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containing solutionsuch as PBS-Tween).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS]and H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generation, e.g.,using a visible spectra spectrophotometer.

D. Vaccination

The present invention contemplates vaccines for use in immunizationembodiments. It is contemplated that proteinaceous compositions that areless effective in promoting VLS or other toxic effects by alterations inone or more (x)D(y), (x)D(y)T and/or flanking sequences may be useful asantigens. In particular embodiments, peptides comprising one or more(x)D(y), (x)D(y)T and/or flanking sequences are contemplated as usefulantigens. Preferably the antigenic material is extensively dialyzed toremove undesired small molecular weight molecules and/or lyophilized formore ready formulation into a desired vehicle. In other embodiments, itis also possible to use toxins lacking one or more active site residues(i.e., a toxoid) as vaccines.

1. Immunomodulators

It is contemplated that immunomodulators can be included in the vaccineto augment the patient's response. Immunomodulators can be included aspurified proteins or their expression engineered into the cells whencells are part of the composition. The following sections list examplesof immunomodulators that are of interest.

a. Cytokines

Interleukins and cytokines, and vectors expressing interleukins andcytokines are contemplated as possible vaccine components. Interleukinsand cytokines, include but not limited to interleukin 1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, β-interferon, α-interferon, γ-interferon, angiostatin,thrombospondin, endostatin, METH-1, METH-2, GM-CSF, G-CSF, M-CSF, tumornecrosis factor, TGFβ, LT and combinations thereof.

b. Chemokines

Chemokines or genes that code for chemokines also may be used as vaccinecomponents. Chemokines generally act as chemoattractants to recruitimmune effector cells to the site of chemokine expression. It may beadvantageous to express a particular chemokine gene in combination with,for example, a cytokine gene, to enhance the recruitment of other immunesystem components to the site of treatment. Such chemokines includeRANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10. The skilled artisan willrecognize that certain cytokines are also known to have chemoattractanteffects and could also be classified under the term chemokines.

The preparation of polyvalent vaccine is generally well understood inthe art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903;4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated hereinby reference. Typically, such vaccines are prepared as injectables.Either as liquid solutions or suspensions: solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. The preparation may also be emulsified. The active immunogenicingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thevaccine may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants whichenhance the effectiveness of the vaccines.

Vaccines may be conventionally administered parenterally, by injection,for example, either subcutaneously, intradermally or intramuscularly.Additional formulations which are suitable for other modes ofadministration include suppositories and, in some cases, oral or nasalformulations. For suppositories, traditional binders and carriers mayinclude, for example, polyalkalene glycols or triglycerides: suchsuppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10%, preferably about 1to about 2%. Oral formulations include such normally employed excipientsas, for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonateand the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations orpowders and contain about 10 to about 95% of active ingredient,preferably about 25 to about 70%.

The polyvalent vaccine of the present invention may be formulated intothe vaccine as neutral or salt forms. Pharmaceutically-acceptable salts,include the acid addition salts (formed with the free amino groups ofthe peptide) and those which are formed with inorganic acids such as,for example, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups may also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges areof the order of several hundred micrograms active ingredient pervaccination. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by subsequent inoculations or other administrations. Theintramuscular route may be preferred in the case of toxins with shorthalf lives in vivo.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection or the like. The dosage of the vaccine willdepend on the route of administration and will vary according to thesize of the host.

Various methods of achieving adjuvant effect for the vaccine includesuse of agents such as aluminum hydroxide or phosphate (alum), commonlyused as about 0.05 to about 0.1% solution in phosphate buffered saline,admixture with synthetic polymers of sugars (Carbopol®) used as an about0.25% solution, aggregation of the protein in the vaccine by heattreatment with temperatures ranging between about 70° to about 101° C.for a 30-second to 2-minute period, respectively. Aggregation byreactivating with pepsin treated (Fab) antibodies to albumin, mixturewith bacterial cells such as C. parvum or endotoxins orlipopolysaccharide components of Gram-negative bacteria, emulsion inphysiologically acceptable oil vehicles such as mannide monooleate(Arecel A) or emulsion with a 20% solution of a perfluorocarbon(Fluosol-DA®) used as a block substitute may also be employed.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the supernatant antigens. The assays may be performed bylabeling with conventional labels, such as radionuclides, enzymes,fluorescents, and the like. These techniques are well known and may befound in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932;4,174,384 and 3,949,064, as illustrative of these types of assays.

2. Adjuvants

Immunization protocols have used adjuvants to stimulate responses formany years. Some adjuvants affect the way in which antigens arepresented. For example, the immune response is increased when proteinantigens are precipitated by alum. Emulsification of antigens alsoprolongs the duration of antigen presentation. Other adjuvants, forexample, certain organic molecules obtained from bacteria, act on thehost rather than on the antigen. An example is muramyl dipeptide(N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), a bacterialpeptidoglycan. The effects of MDP, as with most adjuvants, are not fullyunderstood. MDP stimulates macrophages but also appears to stimulate Bcells directly. The effects of adjuvants, therefore, are notantigen-specific. If they are administered together with a purifiedantigen, however, they can be used to selectively promote the responseto the antigen.

Adjuvants have been used experimentally to promote a generalizedincrease in immunity against unknown antigens (e.g., U.S. Pat. No.4,877,611). This has been attempted particularly in the treatment ofcancer. For many cancers, there is compelling evidence that the immunesystem participates in host defense against the tumor cells, but only afraction of the likely total number of tumor-specific antigens arebelieved to have been identified to date. However, using the presentinvention, the inclusion of a suitable adjuvant into the membrane of anirradiated tumor cell will likely increase the anti-tumor responseirrespective of the molecular identification of the prominent antigens.This is a particularly important and time-saving feature of theinvention.

The present invention contemplates that a variety of adjuvants may beemployed in the membranes of cells, such as tumor cells, resulting in animproved immunogenic composition. The only requirement is, generally,that the adjuvant be capable of incorporation into, physical associationwith, or conjugation to, the cell membrane of the cell in question.

Those of skill in the art will know the different kinds of adjuvantsthat can be conjugated to cellular vaccines in accordance with thisinvention and these include alkyl lysophosphilipids (ALP); BCG; andbiotin (including biotinylated derivatives) among others. Certainadjuvants particularly contemplated for use are the teichoic acids fromGram⁻ cells. These include the lipoteichoic acids (LTA), ribitolteichoic acids (RTA) and glycerol teichoic acid (GTA). Active forms oftheir synthetic counterparts may also be employed in connection with theinvention (Takada et al., 1995).

Hemocyanins and hemoerythrins may also be used in the invention. The useof hemocyanin from keyhole limpet (KLH) is particularly preferred,although other molluscan and arthropod hemocyanins and hemoerythrins maybe employed.

Various polysaccharide adjuvants may also be used. For example, Yin etal., (1989) describe the use of various pneumococcal polysaccharideadjuvants on the antibody responses of mice. The doses that produceoptimal responses, or that otherwise do not produce suppression, asindicated in Yin et al., (1989) should be employed. Polyamine varietiesof polysaccharides are particularly preferred, such as chitin andchitosan, including deacetylated chitin.

A further preferred group of adjuvants are the muramyl dipeptide (MDP,N-acetylmuramyl-L-alanyl-D-isoglutamine) group of bacterialpeptidoglycans. Derivatives of muramyl dipeptide, such as the amino acidderivative threonyl-MDP, and the fatty acid derivative MTPPE, are alsocontemplated.

U.S. Pat. No. 4,950,645 describes a lipophilic disaccharide-tripeptidederivative of muramyl dipeptide which is proposed for use in artificialliposomes formed from phosphatidyl choline and phosphatidyl glycerol. Itis said to be effective in activating human monocytes and destroyingtumor cells, but is non-toxic in generally high doses. The compounds ofU.S. Pat. No. 4,950,645 and PCT Patent Application WO 91/16347, whichhave not previously been suggested for use with cellular carriers, arenow proposed for use in the present invention.

A preferred adjuvent in the present invention is BCG. BCG (bacillusCalmette-Guerin, an attenuated strain of Mycobacterium) and BCG-cellwall skeleton (CWS) may also be used as adjuvants in the invention, withor without trehalose dimycolate. Trehalose dimycolate may be useditself. Azuma et al., (1988) show that trehalose dimycolateadministration correlates with augmented resistance to influenza virusinfection in mice. Trehalose dimycolate may be prepared as described inU.S. Pat. No. 4,579,945.

BCG is an important clinical tool because of its immunostimulatoryproperties. BCG acts to stimulate the reticulo-endothelial system,activates natural killer cells and increases proliferation ofhematopoietic stem cells. Cell wall extracts of BCG have proven to haveexcellent immune adjuvant activity. Recently developed molecular genetictools and methods for mycobacteria have provided the means to introduceforeign genes into BCG (Jacobs et al., 1987; Snapper et al., 1988;Husson et al., 1990; Martin et al., 1990). Live BCG is an effective andsafe vaccine used worldwide to prevent tuberculosis. BCG and othermycobacteria are highly effective adjuvants, and the immune response tomycobacteria has been studied extensively. With nearly 2 billionimmunizations, BCG has a long record of safe use in man (Luelmo, 1982;Lotte et al., 1984). It is one of the few vaccines that can be given atbirth, it engenders long-lived immune responses with only a single dose,and there is a worldwide distribution network with experience in BCGvaccination. An exemplary BCG vaccine is sold as TICE® BCG (OrganonInc., West Orange, N.J.).

In a typical practice of the present invention, cells of Mycobacteriumbovis-BCG are grown and harvested by methods known in the art. Forexample, they may be grown as a surface pellicle on a Sauton medium orin a fermentation vessel containing the dispersed culture in a Dubosmedium (Dubos et al., 1947; Rosenthal, 1937). All the cultures areharvested after 14 days incubation at about 37° C. Cells grown as apellicle are harvested by using a platinum loop whereas those from thefermenter are harvested by centrifugation or tangential-flow filtration.The harvested cells are re-suspended in an aqueous sterile buffermedium. A typical suspension contains from about 2×10¹⁰ cells/ml toabout 2×10¹² cells/ml. To this bacterial suspension, a sterile solutioncontaining a selected enzyme which will degrade the BCG cell coveringmaterial is added. The resultant suspension is agitated such as bystirring to ensure maximal dispersal of the BCG organisms. Thereafter, amore concentrated cell suspension is prepared and the enzyme in theconcentrate removed, typically by washing with an aqueous buffer,employing known techniques such as tangential-flow filtration. Theenzyme-free cells are adjusted to an optimal immunological concentrationwith a cryoprotectant solution, after which they are filled into vials,ampoules, etc., and lyophilized, yielding BCG vaccine, which uponreconstitution with water is ready for immunization.

Amphipathic and surface active agents, e.g., saponin and derivativessuch as QS21 (Cambridge Biotech), form yet another group of preferredadjuvants for use with the immunogens of the present invention. Nonionicblock copolymer surfactants (Rabinovich et al., 1994; Hunter et al.,1991) may also be employed. Oligonucleotides, as described by Yamamotoet al., (1988) are another useful group of adjuvants. Quil A andlentinen complete the currently preferred list of adjuvants. Althougheach of the agents, and the endotoxins described below, are well-knownas adjuvants, these compounds have not been previously incorporated intothe membrane of a target cell, as shown herein.

One group of adjuvants particularly preferred for use in the inventionare the detoxified endotoxins, such as the refined detoxified endotoxinof U.S. Pat. No. 4,866,034. These refined detoxified endotoxins areeffective in producing adjuvant responses in mammals.

The detoxified endotoxins may be combined with other adjuvants toprepare multi-adjuvant-incorporated cells. Combination of detoxifiedendotoxins with trehalose dimycolate is contemplated, as described inU.S. Pat. No. 4,435,386. Combinations of detoxified endotoxins withtrehalose dimycolate and endotoxic glycolipids is also contemplated(U.S. Pat. No. 4,505,899), as is combination of detoxified endotoxinswith cell wall skeleton (CWS) or CWS and trehalose dimycolate, asdescribed in U.S. Pat. Nos. 4,436,727, 4,436,728 and 4,505,900.Combinations of just CWS and trehalose dimycolate, without detoxifiedendotoxins, is also envisioned to be useful, as described in U.S. Pat.No. 4,520,019.

Various adjuvants, even those that are not commonly used in humans, maystill be employed in animals, where, for example, one desires to raiseantibodies or to subsequently obtain activated T cells. The toxicity orother adverse effects that may result from either the adjuvant or thecells, e.g., as may occur using non-irradiated tumor cells, isirrelevant in such circumstances.

E. Pharmaceutical Preparations

Pharmaceutical aqueous compositions of the present invention comprise aneffective amount of one or more IT, VLS inhibitory peptide orpolypeptide, VLS stimulatory peptide or polypeptide and/or cytokinedissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium. The phrases “pharmaceutically or pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to a human. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

The following buffers and reagents are particularly contemplated for usein the preparation of pharmaceutical preparations of the presentinvention: dgRTA, deglycosylated ricin A chain; DMF, dimethylformamide(Pierce, Rockford, Ill.); DTT (Pierce); PBE, 0.05 M sodium phosphate, pH7.5 with 1 mM EDTA; PBES, 0.05 M sodium phosphate, pH 7.5 with variousconcentrations of NaCl (such as 0.1 M, 0.2 M, 0.3 M, 0.4 M and 0.5 MNaCl) and 1 mM EDTA; PBSE, 0.1 M sodium phosphate, pH 7.5 with 0.17 MNaCl and 1 mM EDTA; SMPT,N-succinimidyl-oxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (Pierce).All buffers may be prepared with endotoxin-free distilled water usingenzyme grade salts (Fisher Biotec, Springfield, N.J.).

The ITs, peptides or polypeptides and/or cytokines may be formulated forparenteral administration, e.g., formulated for injection via theintravenous, intramuscular or sub-cutaneous routes, though other routessuch aerosol administration may be used. The preparation of an aqueouscomposition that contains at least one IT, proteinaceous material and/orcytokine as an active ingredient will be known to those of skill in theart in light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980,incorporated herein by reference. Moreover, for human administration, itwill be understood that preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards.

Typically, such compositions can be prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for preparingsolutions or suspensions upon the addition of a liquid prior toinjection can also be prepared; and the preparations can also beemulsified. The compositions will be sterile, be fluid to the extentthat easy syringability exists, stable under the conditions ofmanufacture and storage, and preserved against the contaminating actionof microorganisms, such as bacteria and fungi. It will be appreciatedthat endotoxin contamination should be kept minimally at a safe level,for example, less that 0.5 ng/mg protein.

Although it is most preferred that solutions of ITs, peptide orpolypeptides and/or cytokines be prepared in sterile water containingother non-active ingredients, made suitable for injection, solutions ofsuch active ingredients can also be prepared in water suitably mixedwith a surfactant, such as hydroxypropylcellulose, if desired.Dispersions can also be prepared in liquid polyethylene glycols, andmixtures thereof and in oils. The carrier can also be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, propylene glycol, and liquid polyethylene glycol, and thelike), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants.

The prevention of the action of microorganisms can be brought about byvarious antibacterial ad antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

It is particularly contemplated that suitable pharmaceutical IT, peptideor polypeptide compositions will generally comprise, but are not limitedto, from about 10 to about 100 mg of the desired IT conjugate, peptideor polypeptide admixed with an acceptable pharmaceutical diluent orexcipient, such as a sterile aqueous solution, to give a finalconcentration of about 0.25 to about 2.5 mg/ml with respect to theconjugate, in, for example, 0.15 M NaCl aqueous solution at pH 7.5 to9.0. The preparations may be stored frozen at −10° C. to −70° C. for atleast 1 year.

F. Kits

In still further embodiments, the present invention concerns kits foruse with the IT or vaccination methods described above. Toxins,cytokines or antigenic compositions with reduced VLS promoting or toxiceffects may be provided in a kit. Such kits may be used to combine thetoxin with a specific antibody to produce an IT, provide cytokines withreduced toxicity, or provide antigens for vaccination in a ready to useand storable container. Additionally, peptide inhibitors of VLSproducing sequences or proteinaceous enhancers of extravasation may beincluded in a kit. However, kits including combinations of suchcomponents may be provided. The kits will thus comprise, in suitablecontainer means, a proteinaceous composition with reduced or enhancedVLS promoting activity. The kit may comprise an antibody or IT insuitable container means.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe and/or other container means,into which the at least one protein, polypeptide or peptide may beplaced, and/or preferably, suitably aliquoted. The kits of the presentinvention may include a means for containing at least one antibody, ITand/or any other reagent containers in close confinement for commercialsale. Such containers may include injection and/or blow-molded plasticcontainers into which the desired vials are retained.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Structural Motif for Initiating Vascular Leak Syndrome

This example demonstrates that a three amino acid sequence motif,(x)D(y), in toxins and IL-2 is responsible for damaging vascular ECs.Short (<20 amino acid) (x)D(y) motif-containing peptides from RTA orIL-2 which contained flanking glycines and a cysteine, as well aspeptides with deleted or mutated sequences were generated. Thesepeptides were attached via the cysteine to a mouse IgG1 Mab (RFB4) notreactive with HUVECs. The VLS-inducing ability of this IgG-peptideconjugate (IgG-RTA) in three VLS model systems was compared. The firstwas in vitro damage to human umbilical endothelial cells, (HUVECs)(Soler-Rodriguez et al., 1993); the second was in vivo fluid accompaniedin mouse lungs (Baluna and Vitetta, 1996); and the third was in vivohuman skin xenografts in SCID mice (Baluna and Vitetta, 1998).

Peptide Synthesis. A peptide representing 13 amino acids (residues69-81, SEQ ID NO:1) from RTA, with added N- and C-terminal glycineresidues to improve solubility (Table 3), was synthesized. The peptidescontaining the x(D)y motif were difficult to solubilize even with theadditional three flanking glycines on each end of the peptide. For thisreason, they were conjugated to a soluble carrier protein. The MAb RFB4was chosen because the RFB4-dgRTA is a prototypic IT, and thereforeRFB4-peptides should “mimic” ITs.

An N-terminal cysteine was added to couple the peptide to the RFB4 MAb.Two RTA control peptides (Table 3) were synthesized. A peptide of 9amino acids representing residues 15-23 from IL-2 as well as a controlpeptide (Table 3) was also synthesized. Again, flanking glycines and acysteine were added. All peptides were synthesized on an AppliedBiosystems Model 430A Solid-phase Peptide Synthesizer.

TABLE 3 Peptides from RTA and IL-2¹ Origin Designation Type PeptideSequence RTA LDV+ NativeCysGlyGlyGlySerValThrLeuAlaLeuAspValThrAsnAlaTyrValGlyGlyGly SEQ ID NO:4              69 70  71 72  73 74  75  76  77 78  79  80 81 LDV−Deleted CysGlyGlyGlySerValThrLeuAlaThrAspAlaTyrValGlyGlyGly SEQ ID NO: 5               69  70 71  72  73 77  78  79  80 81 GQT+ MutantCysGlyGlyGlySerValThrLeuAlaGlyGlnThrThrAsnAlaTyrValGlyGlyGly SEQ ID NO:6                 69 70 71  72 73   74 75  76  77 78 IL-2 LDL+ NativeCysGlyGlyGlyGluHisLeuLeuLeuAspLeuGlnMetGlyGlyGly SEQ ID NO: 7             15 16  17 18  19  20  21 22  23 LDU⁻ DeletedCysGlyGlyGlyGluHisLeuLeuGlnMetGlyGlyGly SEQ ID NO: 8               15 1617 18  22  23 ¹Each peptide was conjugated to the mouse MAb, RFB4 asdescribed

Conjugation of the peptides to RFB4. All peptides contained anN-terminal cysteine residue to facilitate conjugation withmaleimide-derivatized RFB4. RFB4 was treated with a 25-fold molar excessof succinimidyl 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate andexcess reagent was removed by gel filtration. The number of maleimidegroups introduced into each molecule of RFB4 was determined by thebacktitration of 2-mercaptoethylamine using Ellman's reagent (Husain andBieniarz, 1994). The derivatized RFB4 was reacted with a 10-fold excessof the SH-peptide at room temperature for 4 hr and excess peptide wasremoved by dialysis against PBS. The maleimide reaction allowed theformation of the IgG1-C—S-peptide conjugate in which the number ofpeptide groups attached was similar to that of free maleimide groups.

As determined by both HPLC and radiolabeling, the RFB4-peptideconjugates (Table 3) contained 6 to 9 maleimide groups per molecule ofIgG1 and these groups formed stable thioether bonds by reaction with thecysteine-containing peptides.

Effect of the RFB4-peptides on the morphology of HUVEC monolayers. Todetermine whether the LDV sequence in RTA and the LDL sequence in IL-2damage HUVECs, monolayers were incubated with different concentrationsof RFB4-RTA-peptides, RFB4-IL-2-peptides or controls. HUVECs wereisolated, cultured and studied microscopically (Baluna et al., 1996;Soler-Rodriguez et al., 1993).

HUVEC monolayers were incubated at 37° C. for 18 hr with 10⁻⁶ MRFB4-LDV⁺, RFB4-LDV⁻, RFB4-GQT, RFB4-LDL⁺, RFB4-LDL⁻ or medium-only andthen examined by phase-contrast microscopy (magnification 20×). Normalmonolayers consisted of highly packed cells with elogated shapes whereasdamaged cells rounded up and detached from the plate. Untreated HUVECsconsisted of tightly packed elongated cells. Treatment with 10⁻⁶ MRFB4-LDV⁺ or RFB4-LDL⁺ caused cell rounding after 2 hr of incubation andthe formation of gaps in the monolayer after 18 hr. Toxic effects onHUVECs were not observed using RFB4-LDV⁻, RFB4-GQT, or RFB4-LDL⁻. Thetoxic effect of RFB4-peptides containing LDV or LDL were dose-dependentand comparable to the effects observed using RFB4-dgRTA (Table 4). Theseresults indicate that the LDV sequence in RTA and its LDL homologue inIL-2 are involved in the EC toxicity of these agents.

TABLE 4 Effect of different concentrations of the RFB4-peptideconstructs on the morphology of HUEC monolayers¹ Concentration (M)² 10⁻⁶10⁻⁷ 10⁻⁸ 0 RFB4-RTA-derived peptides RFB4-LDV+ ++ ++ + − RFB4-LDV− − −− − RFB4-GGT+ − − − − RFB4-IL-2-derived peptides RFB4-LDL+ ++ ++ + −RFB4-LDL− − − − − RFB4-dgRTA ++ + + − RFB4 − − − − ¹HUVECs were grown toconfluence in 96-well tissue culture plates and cells were treated foreighteen hours with different concentrations of RTA-derivedpeptide-constructs in M199 with 2% FCS. ²The morphological changes werescore as “−” no changes, “+” rounding up of cells and “++” disruptionand detachment of cells from the cell monolayer.

In vivo effect of the RFB4-peptides. Although the vascular toxicity ofIL-2 has been observed in experimental animals (Orucevic and Lala, 1995;Puri et al., 1989; Puri and Rosenberg, 1989; Rosenstein et al., 1986),it has been difficult to induce dgRTA-IT-mediated systemicmanifestations of VLS in mice, rats or monkeys (Soler-Rodriguez, 1992).A model has been developed to study the effect of ITs on humanendothelium in vivo by grafting vascularized human skin onto SCID mice,injecting the mice with dgRTA-ITs and measuring fluid accumulation inthe graft as the wet/dry weight ratio (Baluna et al., 1998). Fluidaccumulation in the human skin was measured by weighing punch biopsiesof the skin grafts before and after freeze drying. This model was usedto evaluate the effect of RFB4-LDV⁺, RFB4-GQT⁺, and RFB4-dgRTA in vivo(FIG. 1A).

The fluid accumulation in the lungs of normal SCID mice was alsoevaluated as IL-2 induces fluid accumulation in the lungs of mice(Orucevic and Lala, 1995). The water content of the lungs or skin graftswas calculated as the wet/dry weight ratio.

Although it has been difficult to demonstrate systemic manifestations ofVLS in mice injected with RTA-ITs, vascular leak occurs in human skinxenografts in SCID mice. Increases in the wet/dry weight ratio of thehuman skin grafts after injection of RFB4-LDV⁺ and RFB4-dgRTA were foundbut not after injection of RFB4-GQT⁺. The fluid accumulation in thesexenografts was comparable using either RFB4-dgRTA or RFB4-LDV⁺.Comparable results were obtained using SCID mouse lungs (FIG. 1B). Itshould be noted that although the difference in the Figures may appearsmall, they are statistically significant and consistent with reportsusing IL-2 (Orucevic and Lala, 1995).

Flow cytometric analysis of the binding of dgRTA and RFB4-peptides toHUVECs. The fact that RFB4-LDV⁺ and RFB4-LDL⁺ damage HUVECs, impliesthat these peptides interact with a binding site on HUVECs, although, inthe intact IL-2 or RTA molecules, the (x)D(y) motif may not be theprimary binding site for ECs. To address these issues with toxins, aseries of binding and binding/inhibition studies were carried out.

The proteins were coupled to fluorescein isothiocyanate (FITC) (Sigma,St. Louis, Mo.). 10⁵ HUVECs were washed twice in cold PBS containing 1%bovine serum albumin (BSA) and 0.01% sodium azide (PBS/BSA/AZ),resuspended in 100 ul of the same buffer and incubated withFITC-reagents for 30 minutes on ice in the dark, washed three times withPBS/BSA/AZ, fixed in 0.5 ml of 1% paraformaldehyde PBS/AZ, and analyzedusing a FACScan (Becton Dickinson, Mountain View, Calif.) and theCytoQuest software.

The binding of dgRTA, PE38-lys and RFB4-peptides to HUVECs was examined.10⁵ HUVECs were incubated on ice for 30 min with FITC-reagents in 100 μlPBS/BSA/AZ at varying, concentrations, washed, fixed in 1%paraformaldehyde and analyzed by flow cytometry. The values representthe mean±SD of three experiments using FITC-dgRTA, FITC-PE38-lys, andFITC-carbonic anhydrase as a control. Histograms of flow cytometricanalyses of the binding of dgRTA, PE38-lys and carbonic anhydrase toHUVECs were made. The binding of FITC-RFB4-LDV⁺ (▴), FITC-RFB4-LDV⁻,FITC-RFB4-GQT⁺, FITC-RFB4, FITC-RFB4-LDL⁺, and FITC-RFB4-LDL⁻ were alsoevaluated. Histograms of RFB4-LDV⁺, RFB4-LDL⁺ and RFB4 were also made.The results of this study demonstrated that the 50% of maximal bindingof FITC-dgRTA and FITC-PE38-lys required 0.035 μg and >100 μg/10⁵ cells,respectively, demonstrating that dgRTA has a >3 log higher relativebinding affinity for HUVECs than PE38-lys. This may be due to the factthat the LDV receptor on HUVECs has a lower affinity for homologoussequences in PE38-lys and/or that LDV in RTA, is more exposed. It isalso possible that other non-homologous sequences in RTA (but not inPE38-lys) bind to HUVECs. The difference between the relative bindingaffinity of FITC-dgRTA (0.035 μg/10⁵ cells/100 μl) and FITC-RFB4-LDV⁺(0.5 μg/10⁵ cells/100 μl) was only 2-fold if calculated on molar basis.Since the RFB4-peptide conjugates with deleted or mutated LDV sequencesdid not bind to HUVECs, the (x)D(y) motif is clearly involved in thebinding.

Inhibition of the binding of dgRTA and RFB4-peptides to HUVECs. Toprovide further evidence for the role of the (x)D(y) motif in thebinding of RTA to HUVECs, a series of binding inhibition studies werecarried out. FITC-dgRTA or FITC-RFB4-LDV⁺ at concentrations representing20-50% of maximal binding (0.035 μg/10⁵ cells for dgRTA and 1.0 μg/10⁵cells for RFB4-LDV⁺) were incubated with HUVECs in the presence orabsence of a 100-fold excess of dgRTA (Inland Laboratories, Austin,Tex.), RFB4-LDV⁺, RFB4, Fn (GIBCO Laboratories, Grand Island, N.Y.) orPE38-lys (NCI, Bethesda) for 30 min on ice in the dark. Washed cellswere fixed in 1% paraformaldehyde and analyzed on the FACS.

It was found that the binding of FITC-dgRTA to HUVECs was inhibitedby >90% by dgRTA and by >60% by RFB4-LDV⁺ indicating that the binding ofdgRTA is specific and that it involves, at least in part, the LDVsequence (FIG. 2A). The fact that the homologue-containing PE38-lyscould not inhibit the binding of dgRTA (FIG. 2A) may be due to the factthat its relative affinity for HUVECs is more than three logs lower. Inaddition, dgRTA may have additional non-homologue binding sites forHUVECs, as suggested by the fact that RFB4-LDV⁺ inhibited its binding by60% and not 100%. Furthermore, in the reverse studies, both dgRTA andRFB4-LDV⁺ inhibited the binding of FITC-RFB4-LDV⁺ to HUVECs to a similarextent (FIG. 2B), further indicating that the LDV sequence in RTA isinvolved in binding to HUVECs. Surprisingly, PE38-lys very effectivelyinhibited the binding of FITC-LDV⁺ to HUVECs (FIG. 2B) indicating thatone or more of its LDV homologue sequences can compete with the LDVmotif for binding of an LDV-containing peptide. It is contemplated thatone or more homologue sequences in PE38-lys (GDL-348-350; GDV-430-432;or GDL-605-607) bind to and damage HUVEC. Fn also inhibited the bindingof both FITC-dgRTA (FIG. 2A) and FITC-RFB4-LDV⁺ (FIG. 2B) to HUVECs, butit did so less effectively. In this regard, although Fn also containsthe LDV motif, it has different flanking residues which may play a rolein the availability of its LDV motif.

The data described above demonstrates that peptides containing the LDVmotif in RTA and the LDL motif in IL-2, when attached to the RFB4 MAbspecifically bind to and damage HUVECs in vitro. The IgG-peptideconjugates and the IgG-RTA IT were equally effective in inducingendothelial cell damage and increased vascular permeability in all threemodels.

The LDV sequence in RTA may be responsible for the initiation of eventsleading to VLS-like symptoms in vivo since injection ofRFB4-RTA-peptides containing the native, but not mutated or deleted, LDVsequence caused vascular leak in lungs and in human skin xenografts in amanner analogous to that of the RFB4-dgRTA IT. dgRTA utilizes its LDVsequence, at least in part, to bind to HUVECs since peptides or proteinscontaining this motif inhibited the dose-dependent, saturable binding ofRTA to HUVECs.

The stereoviews of LDV in RTA and LDL in IL-2 indicate that these motifsare partially exposed and should interact with cells. For RTA, this issupported by its dose dependent, saturable binding to HUVECs in vitro.Since the binding of RFB4-LDV⁺ to HUVECs could be partially inhibitednot only by dgRTA but also by proteins containing LDV or LDV-homologues,i.e. Fn and PE38-lys, this further indicates a functional conservationin the (x)D(y) motif in several divergent molecules. Deletions ormutations in this sequence or the use of non-damaging blocking peptidesmay increase the therapeutic index of both IL-2 as well as ITs preparedwith a variety of plant or bacterial toxins.

Example 2 Reduced Pulmonary Vascular Leak in Mice

In this example, it was demonstrated that the enzymatic site or theputative VLS-inducing site in RTA can be mutated without effecting theactivity of the other site. The results showed that an active sitemutant (E177D) induces EC damage and pulmonary vascular leak while oneparticular LDV mutant (L74A) makes an active IT but does not induce thisdamage. Thus, a single amino acid change (L74A) yields an RTA with thedesirable properties of IT activity with reduced vascular damage. Theseresults demonstrate that it is now possible to generate an effectiveRTA-containing IT which does not cause VLS.

Plasmids and mutagenesis. It has been shown that E177 in RTA is one ofseveral amino acids involved in the active site and that an E177D mutanthas greatly reduced enzymatic activity. The pKK223 plasmid with (wt RTAgene) and the pUC 18 plasmid (E177D), both under IPTG-inducible control(O'Hare et al., 1987; Simpson et al., 1995). In addition, from the(wt)RTA construct, the RTA mutants with conserved changes in the LDVsequence were generated. All DNA manipulations were performed usingstandard techniques (Sambrook et al., 1989). Mutations were introducedinto the wt sequence using QuikChange® (Stratagene) and mutagenic primerpairs as shown in Table 5. These mutants included L74A, D75N, D75A, D75Eand V76A.

TABLE 5 Mutants and Primers Designation Amino Acid Sequence¹ DesignationMutagenic Primer Sequences² Wt LeuAlaLeuAspValThrAsnAlaTyrValVal SEQ IDNO: 9 L74a LeuAlaAlaAspValThrAsnAlaTyrValVal SEQ ID NO: 15CTTTCTGTTACATTAGCCGCGGATGTCACCAATGCATATG SEQ ID NO: 10 D75ALeuAlaLeuAlaValThrAsnAlaTyrValVal SEQ ID NO: 16GTTACATTAGCCCTGGCTGTCACCAATGCATATG SEQ ID NO: 11 D75ELeuAlaLeuGluValThrAsnAlaTyrValVal SEQ ID NO: 17CTGTTACATTAGCCCTGGAAGTCACCAATGCATATG SEQ ID NO: 12 D75NLeuAlaLeuAspValThrAsnAlaTyrValVal SEQ ID NO: 18CTGTTACATTAGCCCTGAACGTCACCAATGCATATGTGG SEQ ID NO: 13 V76ALeuAlaLeuAspAlaThrAsnAlaTyrValVal SEQ ID NO: 19GTTACATTAGCCCTGGATGCTACCAATGCATATGTGGTC SEQ ID NO: 14 ¹VLS consensussequence, LDV, is underlined; the active site residue, Tyr, is bold ²Apair of primers corresponding to both complementary strands of thissequence used in the mutagenesis reaction

Expression of RTA in E. coli. Overnight cultures of E. coli strainXL1-Blue freshly transformed with either plasmid in Terrific Broth(Sambrook et al., 1989) containing 100 μg/mL ampicillin at 37° C. wereused to inoculate (1%) 500 mL of the same media (in 2 L flasks).Cultures were grown at 30° C. with vigorous shaking until they hadreached an OD₆₀₀ of 0.6 to 0.8. Expression was induced using 0.3 mM IPTGand the cultures allowed to grow overnight (˜15 h). Scaled up expressionwas carried out in a 5 L fermentor (New Brunswick Scientific, Edmon,N.J.) with the same media and inoculum as above. Cultures were grownwith agitation of 400 rpm and airflow of 4.0 L/min at 37° C. until OD₆₀₀of 0.5. The cultures were slowly cooled to 30° C., induced with 1.0 mMIPTG, and grown 16-18 h with agitation of 250 rpm and airflow of 2.5L/min. Cells were harvested and resuspended in 10 mL PBS (50 mMphosphate-buffered saline, pH 7.0) and lysed by sonication (six30-second bursts) or by passage through a French Press (SpectronicInstruments). Cell debris was removed by centrifugation at 15K rpm for20 min; supernatants were filtered (0.2 μm) and stored at −20° C. untilpurification.

Radioimmunoassay (RIA) and bioassay of expressed RTA. The yield ofexpressed RTA was assayed using a solid phase RIA. In this assay wellsof microtiter plates are coated with an affinity purified rabbitanti-RTA. Plates are washed and blocked with BSA. Dilutions of purifiedrRTA (standard curve) or of sample are added for 2 hrs at 25° C. Platesare washed and 100,000 cpm of ¹²⁵S affinity purified rabbit anti-RTA isadded to each well for 1 hr at 25° C. Plates are washed. Wells are cutout and counted on a gamma counter. Concentration of rRTA in the samplesare determined from the standard curve. Briefly, 96-well plates werecoated with polyclonal rabbit anti-RTA, blocked with fetal calf serum,and serial dilutions of either the dgRTA or wt RTA standard or dilutionsof the rRTA-containing E. coli lysates were added. Wells were washed andbound RTA was detected using ¹²⁵I-labelled rabbit anti-RTA. The amountsof RTA in the E. coli extracts were determined from the standard curve.

Purification of rRTAs. rRTAs were purified from the bacterial lysates byion-exchange chromatography on CM-Sepharose fast flow (Pharmacia). pH6-9 proteins were eluted using a 0-300 mM NaCl gradient. Pooledfractions, comprising the main protein peak typically contained 50-80%rRTA. This pool was further purified by chromatography on Blue-SepharoseCL-4B; bound rRTA was eluted with 1 M NaCl (Ghetie et al., 1991). TheRTA preparations were concentrated to 3-4 mg/mL and stored at −20° C. in50% glycerol. Purified recombinant (r) RTA preparations were evaluatedby sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)and were >90% pure. The enzymatic activity of wt rRTA, dgRTA and themutant rRTAs was determined using a cell-free rabbit reticulocyte assay(Press et al., 1988).

Preparation of RFB4-RTA. The murine Mab, RFB4 (anti-human-CD22) waschemically conjugated to rRTAs and dgRTA usingN-succinimidyl-oxycarbonyl-α-methyl-α (2-pyridyldithio) toluene (SMPT,Pierce) and purified as described previously for dgRTA (Ghetie et al.,1991; Knowles and Thorpe, 1987). The enzymatic activity of the RTA inthe ITs was tested in the cell-free rabbit reticulocyte assay, followingreduction (Press et al., 1988).

In vitro cytotoxicity assays. The cytotoxic activities of the differentRFB4-containing-ITs were determined using CD22⁺ Daudi cells and[3H]-leucine incorporation as described previously (Ghetie et al.,1988). The concentration of IT which reduced [³H]-leucine incorporationby 50% relative to an untreated control culture was defined as the IC₅₀.

As shown in Table 6, when tested in the reticulocyte assay, the wt RTAand dgRTA had very similar activities although the wt RTA was slightlymore active. Following coupling to the RFB4 Mab, dgRTA was approximately3-fold less toxic than wt RTA. In contrast, the E177D rRTA and theRFB4-E177D IT were 3200 fold and 560 fold less enzymatically activite,respectively, in the reticulocyte assay and greater than 5×10⁷ fold lesstoxic in the Daudi cell assay (Table 5 and FIG. 3). RFB4-E177D, whichbound to Daudi cells as effectively as RFB4-wtRTA, was virtuallyinactive as an IT.

TABLE 6 The Enzymatic Activity of rRTAs and ITs prepared with theserRTAs Cell Free Daudi Reticulocyte Cytotoxicity Fold decrease AssayAssay in activity of Fold decrease Fold decrease RTA vs in activity vsin activity vs RTA wt RTA wt RFB4-RTA wt RFB4-RTA wt —^(a) —^(b) —^(c)DgRTA 1.3 ± 0.6 (8)^(d) 3.0 ± 1.1 (6) 4.4 ± 1.6 (19) L74N^(e) 1.5 ± 0.4(3) 1.2 ± 0.8 (3) 9.1 ± 5.3 (4) D75N^(e) 13.5 ± 6.6 (4) 5.5 ± 1.4 (4)660 ± 270 (3) D75A^(e) 3.1 ± 1.7 (3) 4.0 ± 2.8 (4) 370 ± 240 (4)D75E^(e) 5.1 ± 2.1 (3) 12.2 ± 10.3 (5) 260 ± 200 (5) V76A^(e) (inprogress) 2.7 ± 1.6 (2) 5.5 ± 3.2 (4) E177D^(e) 3200 ± 1100 (3) 560 ±450 (5) 5.0 × 10⁷ (8) ^(a)In 16 studies the IC₅₀ was 1.4 ± 0.7 × 10⁻¹¹M^(b)In 18 studies the IC₅₀ was 6.4 ± 1.8 × 10⁻¹²M ^(c)In 18 studies theIC₅₀ was 1.6 ± 0.9 × 10⁻¹³M ^(d)Number of studies using 3 differentpreparations of each mutant ^(e)Refers to the RTA mutation in the LDVsequence or the active site (E177)

With regard to the LDV mutants, as compared to wt RTA, L74A was 1.2-1.5less active in the reticulocyte assay and 6-9-fold less active as an ITin the Daudi cell cytotoxicity assay (Table 6 and FIG. 3). As comparedto dgRTA, the L74A IT was 4-5-fold less active in the Daudi assay.Similar decreases were observed using V76A (Table 6). In contrast toboth the L74A and V76A mutants, D75A, D75E and D75N were 3-13-fold lessactive in the reticulocyte assay, and >200 fold less active as ITs inDaudi cell cytotoxicitv assays. This result indicated that D75 may beparticularly important for internalization, for routing, or forintracellular stability of the RTA in Daudi cells. As compared to thewtRTA, L74A and V76A formed the most active IT.

Vascular toxicity of RFB4-rRTA. As a first step in evaluating theability of ITs prepared with mutants RTAs to induce vascular damage, aseries of in vitro studies using IIUVECs were conducted. For in vitroassays, the effect of RFB4-rRTA on the morphology of HUVECs monolayerswas tested as described previously (Baluna et al., 1996).

As shown in FIG. 4, the ITs containing wtRTA, dgRTA or the enzymaticallyinactive E177D RTA damaged HUVECs. Since E177D contains LDV, but has adisrupted active site and is inactive as an IT, this indicated that theactive site of RTA and the putative VLS-inducing site (LDV) of RTA aredistinct and that enzymatic activity does not appear to be the mostimportant feature for the ability of RTA to damage HUVECs. In contrast,when ITs containing mutations in L, D or V (but not the active enzymaticsite) were tested on HUVECs, only the IT containing V76A damaged thesecells. Taken together with the results of the Daudi cell studies, theonly IT which was active in the Daudi cell cytotoxicity assay andinactive in the HUVEC assay contained L74A. In contrast, ITs preparedwith the D75 mutants had greatly reduced activity in the Daudicytotoxicity assay even though there were not toxic to HUVECs.Conversely, the IT prepared with E76A, was active on Daudi cells, butdamaged HUVECs. Of the ITs prepared with the 5 LDV mutants, the L74Amutant appeared to contain both of the desired properties in vitro.

For in vivo assays, the effect of RFB4 ITs was determined in theSCID/Daudi tumor model (Ghetie et al., 1992). Female SCID mice wereinjected I.V. (lateral tail vein) with 5×10⁶ Daudi cells on day zero.ITs were injected I.V. on days 1, 2, 3 and 4. Groups of 5 mice were usedfor each treatment and studies were repeated. Treatment groups received(1) no treatment (control); (2) RFB4-dgRTA 40% of the LD50 or 60μg/mouse; (3) RFB4-wt RTA, 40% of the LD50 or 80 μg; (4) RFB4 E177D, 40%of the LD50 or 400 μg/mouse; (5) RFB4 L74A, 40% of the LD50 or 200μg/mouse; (6) RFB4 V76A. Mice were followed and sacrificed when theparalysis of their hind legs occurred. Pulmonary vascular leak inIT-injected SCID mice was evaluated as described (Baluna et al., 1999).The water content of the lungs was calculated as the wet/dry weightratios of lungs removed from mice injected with 10 ug 1 T/g of mouseweight.

Unlike humans, mice injected with RTA-containing ITs do not manifestsystemic VLS in terms of weight gain and edema but they do losesignificant weight and show pulmonary vascular leak. SCID mice wereinjected with the ITs prepared with the mutant rRTAs and monitored bothweight loss and pulmonary vascular leak. As shown in FIG. 5, when micewere injected with 10 ug IT/g of body weight, both weight loss (A) andpulmonary leak (B) were observed using the ITs prepared with dgRTA,wtRTA or V76A. In contrast, the ITs containing L74A D75A, D75N or D75Edid not induce weight loss or pulmonary leak. At this dose, the RFB4-E177D IT did not cause weight loss but did induce pulmonary leak. Theseresults further demonstrated that the IT containing L74A RTA possessesthe desired properties. It is contemplated that weight loss is relatedprimarily to the LDV sequence but that severe weight loss, as seen inthe IT prepared with wt RTA, maybe related to both an active site andthe LDV sequence of the RTA.

When coupled to RFB4, an active site mutant, E177D, induced EC damageand vascular damage in vivo although RFB4-E177D was 10⁷-fold less activethan RFB4-wt RTA in vitro (i.e., it was inactive). A single amino acidchange in RTA (L74A) resulted in the expression of a highly active RTAenzyme which made an effective IT both in vitro and in vivo. As comparedto RFB4-dgRTA which have been used in mice and patients, RFB4-L74A wasonly 5-fold less active. At the same dose as RFB4-wt RTA, RFB4-L74A didnot damage ECs or cause weight loss in mice. As an IT, it had an LD₅₀which was 20-fold higher than that of an IT prepared with wt RTA,indicating that much higher doses should be safe in vivo. In contrast toRFB4-L74, ITs prepared with mutants containing alterations in D75,although enzymatically active, performed very poorly in the Daudicytotoxicity assay. Hence D75 may be involved in internalization,intracellular routing or intracellular stability of the RTA.Additionally, a V76 mutant was both enzymatically active and active asan IT but also induced both EC damage and vascular damage.

Taken together, these studies clearly demonstrate that L74A is adesirable RTA mutant. Because RFB4-L74A has a much higher LD₅₀ in mice,it will now possible to refine dose regimens to determine whether thetherapeutic window has been widened.

Example 3 Production and Purification of dgRTA

Inland Laboratories has formally produced dgRTA. However, the followingexample describes a procedure for producing dgRTA for use in the presentinvention.

A powdered acetone extract of castor beans is the starting material.Ricin is extracted from this powder, the ricin is deglycosylated,separated into A and B chains, and the dgRTA is purified.

Bulk raw material, powdered acetone extract of castor beans, can bepurchased from Sigma Bulk Chemical Sales. The material arrives inplastic bottles which have a 1.0 L capacity and contain 200 grams ofpowder. The bottles are stored in a locked cabinet until the procedureis initiated.

Table 7 lists the composition of the buffers and solutions needed forthe procedure and Table 8 lists the specifications for the buffers andsolutions. Table 9 lists the equipment used for the production andpurification of dgRTA and Table 10 provides a summary of the steps andcolumns used in the procedure.

TABLE 7 COMPOSITION OF BUFFERS AA 200 mM acetic acid adjusted to pH 3.5with 1 M NaOH BB 50 mM boric acid adjusted to pH 8.0 with 1 Mconcentrated NaOH BB + 1 M NaCl BB + 1 M NaCl, pH 8.0 BB + 0.2 M BB +0.2 M galactose, pH 8.0 galactose Deglycosylation AA +40 mM NalOQ, +80mM NaCNBH3, pH 3.5 buffer PBS 50 mM H2NaPO4 + 150 mM NaCl adjusted to pH7.2 with 1 M NaOH Reducing BB BB+4% mercaptoethanol, pH 8.0 Tris 2 MTris-HCl, pH 10.8

TABLE 8 BUFFER SPECIFICATIONS Buffer Sterility PH Conductivity EndotoxinAA Sterile 3.5 0.9 mΩ Zero BB Sterile 8.0 5.0 mΩ Zero BB + 1 M NaClSterile 8.0 60 mΩ Zero BB + 0.2 M Sterile 8.0 4.6 mΩ Zero galactoseDeglycosylation Sterile 3.5 8.0 mΩ Zero buffer PBS Sterile 7.2 11.5 mΩZero Reducing BB Sterile 8.0 5.0 mΩ Zero Tris Sterile 10.8 25 mΩ Zero

TABLE 9 EQUIPMENT Amicon concentrator, Model #CH2 Autoclave, ModelHirayama Biological safety cabinet (hood), Model NuAire #NU-425-400Circulating shaker, Model Fisher #631 1 μ Centrifuge; Model, Jovan#GR-412 Centrifuge tubes description, IEC 750 ml Gravity ConnectionOwen, Model Precision Negative pressure refrigerator (a negativepressure, refrigerated, chromatography box), Model EJS #CR-84 Pharmaciaspectro ultrospec III Technicloth TX 609 (from Tex Wipe) Whatmann 90377A(1.0 p.m) capsule filter

TABLE 10 COLUMN SPECIFICATIONS EQUILIBRA- SIZE BED TION PROTEIN VOLUMEELUTION COLUMN (CM) VOLUME BUFFER LOADED LOADED BUFFER FLOW RATEAcid-Treated 25 × 7.36 L BB ~100,000 ODU   4 L BB + 0.2 M 4.5 L/hSepharose 4B 15 galactose Sephacryl S- 25 × 44.2 L AA  ~7,000 ODU 1.5 LNone 4.5 L/h 200 90 Columns run 11 ×  2.8 L BB 8,000 mg ricin 3.5 LReducing 4.5 L/h in tandem: 30 toxin (RCA2) BB DEAE- 25 × 14.7 LSepharose 30 And Acid-Treated Sepharose 4B Blue 25 × 7.36 L BB 3,000 mgdgA  30 L BB + 1 M 1.5 L/h Sepharose 15 NaCl CL-4B Aslalofetuin- 11 × 2.8 L BB 2,000 mg dgA  10 L None 1.5 L/h Sepharose 30 *All columns arekept in the negative refrigerator at 4° C.

Extraction of bulk ricin: Each extraction batch may consist of twobottles of the castor bean powder. These are opened in the biologicalsafety cabinet (hood), and 1.0 L of PBS is added to each bottle. Eachbottle is capped with the original lid, placed on the circular shaker inthe hood, and shaken for 1 hour at 200 cycles/minute, at roomtemperature (RT). The bottles are placed at 4° C. for 30 min to allowfor sedimentation of particulates. The supernatant from each bottle ispoured into an intermediate vessel which has a cover and spout, and fromthat vessel, the liquid is poured into 750 ml plastic centrifugebottles, both steps being performed in the hood. The centrifuge bottlesare capped and wiped clean with moist paper towels before they areremoved from the hood. The centrifuge bottles are placed in carriers inthe centrifuge and centrifuged at 3,700 rpm for 20 minutes at 4° C. Thecentrifuge bottles are removed, taken to the hood and the supernatantsare decanted and filtered through Technicloth TX609 paper into a 6 LErlenmeyer flask.

A second extraction is performed. The sediment in the two factorybottles is resuspended with 1.0 L PBS and the extraction procedureincluding 1 h of shaking at RT followed by centrifugation and filtrationis repeated.

Purification of ricin: All of the following procedures are performed inthe negative pressure chromatography box at 4° C.

Both the first and second extractions are pooled and filtered through aWhatman 90377A (1.0 pm) capsule filter. The absorbance at 280 nm isdetermined and the total protein extracted from the powder is calculatedand recorded.

The clarified supernatant is then pumped from the flask onto theacid-treated Sepharose 4B column. The unbound fraction is collected,autoclaved and discarded. The bound fraction is eluted with BB+0.2 Mgalactose. The eluate is collected directly into a CH2 Amiconconcentrator and concentrated to 1.5 L. The concentrate is transferredto a flask and the volume is measured. An aliquot is used to measure theabsorbance at 280 nm and the total protein in ODU is recorded.

The protein is pumped from the flask onto the Sephacryl S-200 column,equilibrated with AA. Separation of peak 1 (RCA1=ricin agglutinin) andpeak 2 (RCA2=ricin toxin) is determined by the lowest absorbance at 280nm between peak 1 and peak 2 (see FIG. 7).

The first peak contains ricin agglutinin and is discarded. The secondpeak contains ricin, it is collected directly into the Amiconconcentrator and concentrated to 2.5 mg/ml. The volume, OD 280 and totalamount of protein in mg is recorded. The ricin is pumped from theconcentrator into a capped vessel, which is then wiped with moist papertowels and removed from the chromatography refrigerator.

Deglycosylation of ricin: The vessel containing the ricin solution isopened in the hood. An equal volume of deglycosylation buffer is added.The vessel is capped, wiped, and placed in the chromatographyrefrigerator for a 4 h incubation at 4° C. The vessel is then placed inthe hood, opened, and glycerol is added to a final concentration of 1%in order to stop the reaction. The vessel is again capped, wiped, pldcedin the chromatography refrigerator and kept overnight at 4° C.

Separation of ricin A and B grains and purification of dgRTA: Afterovernight incubation, the vessel is removed from the refrigerator andplaced in the hood. The vessel is opened and Tris is added until the pHreaches 8.0. The neutralized ricin solution is pumped into 2 columnsconnected together in tandem in the chromatography refrigerator. Thefirst column contains DEAF-Sepharose and the second containsacid-treated Sepharose 4B. Both columns ate equilibrated with BB. Thepurpose of the DEAE-Sepharose column is to bind endotoxin. Once theabsorbance at 280 nm of the effluent from the acid-treated Sepharose 413column falls to 0.05 ODU (end of peak 1, FIG. 8), the ricin has gonethrough the DFAE Sepharose column and into the acid-treated Sepharose 4Bcolumn, where it is bound.

Peak 1 represents a small amount of dgRTA that does not bind toacid-treated Sepharose 4B. The columns are then separated and reducingBB is pumped into the acid-treated Sepharose 4B column until theabsorbance at 280 nm equals the absorbance of the reducing BB (0.12ODU). The pump is then stopped and the column is incubated for 4 hoursat 4° C. During this time, the S—S bond between the ricin A and B chainsis reduced which results in the separation of the two chains.

The ricin B chain remains bound to the acid-treated-Sepharose 413column. The column is then washed with reducing BB and the free dgRTA iscollected (peak 2, FIG. 8). The collection is stopped when theabsorbance at 280 nm returns to the absorbance of reducing BB. The OD280 of the collected effluent (dgRTA solution) is measured and thevolume and protein concentration in ODU is recorded.

The acid-treated Sepharose 4B column is eluted with BB+0.2 M galactoseand the eluted ricin B chain is discarded.

The dgRTA solution in reducing BB is then pumped over a column ofBlue-Sepharose CL-4B equilibrated with BB and held at 4° C. The columnis washed with BB until the absorbance at 280 nm drops to baseline. Thecolumn is then washed with BB+0.2 M galactose until the small peak iseluted (Peak 2) and the absorbance at 280 nm returns to the baselinevalue. This procedure is applied to remove all traces of whole ricintoxin (RCA2) or B chain that can interact with the Sepharose matrix.dgRTA is eluted from the Blue-Sepharose column with BB+1 M NaCI as shownin FIG. 9, Peak 3 and the volume and absorbance at 280 nm is measuredand recorded.

The eluate is then pumped onto the Asialofetuin-Sepharose columnequilibrated with BB and held at 4° C. The nonbound fraction, startingwhen the absorbance is over 0.05 ODU and ending when it returns 0.05ODU, as shown in FIG. 10, is diafiltered in the Amicon concentrator todecrease the NeCI concentration to 0.25 M and to increase the proteinconcentration to 2 mg/ml. DTT is then added to a final concentration of10 mM and the solution is filtered through a 0.22 μm filter. The reduceddgRTA solution is mixed with an equal volume of glycerol and stored at−20° C. The column is eluted with BB+0.2 M galactose and the eluate isdiscarded.

Testing and specifications: Table 11 lists the tests and specificationsfor dgRT.

TABLE 11 Tests and Specifications for dgRTA TEST MATERIAL TESTSPECIFICATION DgRTA Protein concentration 2-3 mg/ml Sterility SterileEndotoxin by LAL <5 EU/mg Purity by SDS gel >95% of protein @28-33 KdaMW peak Con A binding <20% bound IC₅₀ (reticulocyte assay) 1-5 × 10¹¹ MIC₅₀ (Daudi cell assay) 05-5 × 10⁷ M LD₅₀ (BALB/c mice) 15-30 μg/g

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold    Spring Harbor, N.Y., 1988.-   Atherton et al., Biol. of Reproduction, 32, 155-171, 1985.-   Azuma et al., “Correlation between augmented resistance to influenza    virus infection and histological changes in lung of mice treated    with trehalose-6,6′-dimycolate,” J Biol Response Mod. 7(5):473-482,    1988.-   Baluna and Vitetta, “An in vivo model to study immunotoxin-induced    vascular leak in human tissue,” J. Immunother., 1999 22(1):41-47,    1999.-   Baluna and Vitetta, “Vascular leak syndrome: A side effect of    immunotherapy,” Immunopharmacology, 37:117-132, 1996.-   Baluna et al., “Fibronectin inhibits the cytotoxic effect of ricin A    chain on endothelial cells,” Int. J. Immunopharm., 18:355-361, 1996.-   Berberian et al., Science, 261:1588-1591, 1993.-   Blobel and White, “Structure, function and evolutionary relationship    of proteins containing a disintegrin domain,” Curr. Opin. Cell    Biol., 4:760-765, 1992.-   Cleary et al., Trends Microbiol., 4:131-136, 1994.-   Clements et al., J. Cell Sci., 107:2127-2135, 1994.-   Collins et al., Proc. Natl. Acad. Sci. USA, 85:7709-7713, 1988.-   Coulson et al., Proc. Natl. Acad Sci. U.S.A, 94:5389-5394, 1997.-   Cunningham et al., “High-resolution epitope mapping of hGH-receptor    interactions by alanine-scanning mutagenesis,” Science    244(4908):1081-1085, 1989.-   De Jager et al., “Current status of cancer immunodetection with    radiolabeled human monoclonal antibodies” Semin Nucl Med 23(2):    165-179, 1993.-   Dholakia et al., J. Biol. Chem., 264, 20638-20642, 1989.-   Doolittle M H and Ben-Zeev O, “Immunodetection of lipoprotein    lipase: antibody production, immunoprecipitation, and western    blotting techniques” Methods Mol Biol., 109:215-237, 1999.-   Downie et al., Am. J. Respir. Cell Molec. Biol., 7:58-65, 1992.-   Dubos et al., 1947.-   Dutcher et al., J. Clin.Oncol., 9:641-648, 1991.-   Engert et al., In: Clinical Applications of Immunotoxins, Frankel    (ed.), 2:13-33, 1997.-   Freifelder, Physical Biochemistry, Second Edition, pages 238-246-   Gefter et al., Somatic Cell Genet. 3:231-236, 1977.-   Ghetie et al., “The GLP large scale preparation of immunotoxins    containing deglycosylated ricin A chain and a hindered disulfide    bond,” J. Immunol Methods, 142(2):223-230, 1991.-   Ghetie et al., Cancer Res. 48:2610, 1988.-   Goding, In: Monoclonal Antibodies: Principles and Practice, 2d ed.,    Orlando, Fla., Academic Press, pp. 60-61, 65-66, 71-74, 1986.-   Greenspoon et al., Int. J. Pept. Res., 43:417-424, 1994.-   Gulbis B and Galand P, “Immunodetection of the p21-ras products in    human normal and preneoplastic tissues and solid tumors: a review”    Hum Pathol 24(12):1271-1285, 1993.-   Halling et al., “Genomic cloning and characterization of a ricin    gene from Ricinus communis,” Nucleic Acids Res. 13(22):8019-8033,    1985.-   Huang, Cellular and Molecular Life Sciences, 54:527-540, 1998.-   Hunter et al., “Adjuvant activity of non-ionic block copolymers. IV.    Effect of molecular weight and formulation on titre and isotype of    antibody,” Vaccine. 9(4):250-256, 1991.-   Husain and Bieniarz, Bioconjug. Chem., 5:481-490, 1994.-   Husson et al., “Gene replacement and expression of foreign DNA in    mycobacteria,” J Bacteriol. 172(2):519-524, 1990.-   Inouye et al., “Up-promoter mutations in the Ipp gene of Escherichia    coli,” Nucl. Acids Res., 13:3101-3109, 1985.-   Jackson et al., J. Med. Chem., 40:3359-3368, 1997.-   Jacobs et al., “Introduction of foreign DNA into mycobacteria using    a shuttle phasmid,” Nature, 327(6122):532-535, 1987.-   Kang et al., Science, 240:1034-1036, 1988.-   Khatoon et al., Ann. of Neurology, 26, 210-219, 1989.-   King et al., J. Biol. Chem., 269, 10210-10218, 1989.-   Knowles, P. P. and Thorpe, P. E. Anal. Biochem., 160:440, 1987.-   Kohler and Milstein, “Continuous cultures of fused cells secretaring    antibody of predefined specificity,” Nature, 256:495-497, 1975.-   Kohler et al., Methods Enzymol., 178:3, 1989.-   Kreier et al., Infection, Resistance and Immunity, Harper & Row, New    York, (1991)).-   Lamb et al., “Nucleotide sequence of cloned cDNA coding for    preproricin,” Eur J Biochem, 148(2):265-270, 1985.-   Lazarus and McDowell, “Structural and functional aspects of    RGD-containing protein antagonists of glycoprotein IIb-IIIa,” Curr.    Opin. Cell Biol., 4:438-445, 1993.-   Lenert et al., Science, 248:1639-1643, 1990.-   Li et al., Proc. Natl. Acad. Sci. USA, 92:9308-9312, 1995.-   Lotte et al., “BCG complications. Estimates of the risks among    vaccinated subjects and statistical analysis of their main    characteristics,” Adv Tuberc Res. 21:107-193, 1984.-   Lu et al., J. Biol. Chem., 271:289-294, 1996.-   Luelmo F., “BCG vaccination,” Am Rev Respir Dis. 125(3 Pt 2):70-72,    1982.-   Maeda et al., Biochem. Biophys. Res. Commun., 241:595-598, 1997.-   Makarem and Humphries, Biochemical Society Transactions,    19:380S-382S, 1991.-   Martin et al., “Transposition of an antibiotic resistance element in    mycobacteria,” Nature, 345(6277):739-743, 1990.-   McLane et al., Proc. Soc. Exp. Biol. Med., 219:109-119, 1998.-   Mlsna et al., Protein Sci., 2:429-435, 1993.-   Munishkin and Wool, J. Biol. Chem., 270:30581-30587, 1995.-   Nakamura et al., In: Enzyme Immunoassays: Heterogeneous and    Homogeneous Systems, Chapter 27, 1987.-   Nowlin et al., J. Biol. Chem., 268:20352-20359, 1993.-   O'Hare et al., Febs Lett., 216(1):73-78, 1987.-   Orucevic and Lala, J. Immunother., 18:210-220, 1995.-   O'Shannessy et al., J. Immun. Meth., 99, 153-161, 1987.-   Owens & Haley, J. Biol. Chem., 259:14843-14848, 1987.-   PCT Patent Application WO 91/16347-   Potter& Haley, Meth. in Enzymol., 91, 613-633, 1983.-   Press et al., J. Immun. 141:4410, 1988.-   Puri and Rosenberg, Cancer Immunol. Immunother., 28:267-274, 1989.-   Puri et al., Cancer Res., 49:969-976, 1989.-   Rabinovich et al., “Vaccine technologies: view to the future,”    Science, 265(5177):1401-1404, 1994.-   Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing    Company, 1980.-   Rosenberg et al., N. Engl. J. Med., 316:889-897, 1987.-   Rosenstein et al., J. Immunol., 137:1735-1742, 1986.-   Rosenthal, 1937.-   Sambrook et al., In: Molecular Cloning: A Laboratory Manual, Vol. 1,    Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Ch.    7, 7.19-17.29, 1989.-   Sasso et al., J. Immunol., 142:2778-2783, 1989.-   Sausville and Vitetta, In: Monoclonal Antibody-Based Therapy of    Cancer, Grossbard (ed.), 4:81-89, 1997.-   Shorki et al., J. Immunol., 146:936-940, 1991.-   Silvermann et al., J. Clin. Invest., 96:417-426, 1995.-   Simpson et al., Eur. J Biochem., 232:458-463, 1995.-   Snapper et al., “Lysogeny and transformation in mycobacteria: stable    expression of foreign genes,” Proc Natl Acad Sci USA.    85(18):6987-6991, 1988.-   Soler-Rodriguez et al., “Ricin A-chain and ricin A-chain    immunotoxins rapidly damage human endothelial cells: implications    for vascular leak syndrome,” Exp. Cell Res., 206:227-234, 1993.-   Soler-Rodriguez et al., Exp. Cell Res., 206:227-234, 1993.-   Soler-Rodriguez et al., Int. J Immunopharm., 14(2):281-291, 1992.-   Takada et al., “Molecular and structural requirements of a    lipoteichoic acid from Enterococcus hirae ATCC 9790 for    cytokine-inducing, antitumor, and antigenic activities,” Infect    Immun. 63(1):57-65, 1995.-   Tselepis et al., J. Biol. Chem., 272:21341-21348, 1997.-   U.S. Pat. No. 3,791,932-   U.S. Pat. No. 3,817,837-   U.S. Pat. No. 3,850,752-   U.S. Pat. No. 3,939,350-   U.S. Pat. No. 3,949,064-   U.S. Pat. No. 3,996,345-   U.S. Pat. No. 4,174,384-   U.S. Pat. No. 4,275,149-   U.S. Pat. No. 4,277,437-   U.S. Pat. No. 4,366,241-   U.S. Pat. No. 4,435,386-   U.S. Pat. No. 4,436,727-   U.S. Pat. No. 4,436,728-   U.S. Pat. No. 4,472,509-   U.S. Pat. No. 4,505,899-   U.S. Pat. No. 4,505,900-   U.S. Pat. No. 4,520,019-   U.S. Pat. No. 4,578,770-   U.S. Pat. No. 4,579,945-   U.S. Pat. No. 4,596,792-   U.S. Pat. No. 4,599,230-   U.S. Pat. No. 4,599,231-   U.S. Pat. No. 4,601,903-   U.S. Pat. No. 4,608,251-   U.S. Pat. No. 4,664,911-   U.S. Pat. No. 4,682,195-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 4,792,447-   U.S. Pat. No. 4,866,034-   U.S. Pat. No. 4,877,611-   U.S. Pat. No. 4,938,948-   U.S. Pat. No. 4,950,645-   U.S. Pat. No. 5,021,236-   U.S. Pat. No. 5,045,451-   U.S. Pat. No. 5,196,066-   U.S. Pat. No. 5,578,706-   U.S. Pat. No. 5,686,072-   Vial and Descotes, Drug Safety, 7:417-433, 1992.-   Vitetta et al., Immunol. Today, 14:252-259, 1993.-   Wayner and Kovach, J. Cell Biol., 116:489-497, 1992.-   Yamamoto et al., 1988.-   Yeh et al., Blood, 292:3268-3276, 1998.-   Yin et al., 1989.

1. A modified ricin toxin A-chain comprising an amino acid substitutionof one or more of Leu-Asp-Val at amino acid residues 74-76 as set forthin SEQ ID NO:1, wherein the induction of vascular leak syndrome by themodified ricin toxin A-chain is reduced as compared to the native ricintoxin A-chain.
 2. The modified ricin toxin A-chain of claim 1,comprising an amino acid substitution of the leucine at amino acidresidue 74 as set forth in SEQ ID NO:1.
 3. The modified ricin toxinA-chain of claim 2, wherein the leucine at amino acid residue 74 as setforth in SEQ ID NO:1 is substituted with a phenylalanine, cysteine,methionine, alanine, threonine, serine, tryptophan, tyrosine, proline,histidine, glutamic acid, glutamine, aspartic acid, asparagine, lysine,or arginine.
 4. The modified ricin toxin A-chain of claim 3, wherein theleucine at amino acid residue 74 as set forth in SEQ ID NO:1 issubstituted with alanine.
 5. The modified ricin toxin A-chain of claim1, comprising an amino acid substitution of the aspartate at amino acidresidue 75 as set forth in SEQ ID NO:1.
 6. The modified ricin toxinA-chain of claim 5, wherein the aspartate at amino acid residue 75 asset forth in SEQ ID NO:1 is substituted with an isoleucine, valine,leucine, phenylalanine, cysteine, methionine, alanine, glycine,threonine, serine, tryptophan, tyrosine, proline, histidine, glutamicacid, glutamine, asparagine, lysine, or arginine.
 7. The modified ricintoxin A-chain of claim 6, wherein the aspartate at amino acid residue 75as set forth in SEQ ID NO:1 is substituted with glutamine.
 8. Themodified ricin toxin A-chain of claim 1, comprising an amino acidsubstitution of the valine at amino acid residue 76 as set forth in SEQID NO:1.
 9. The modified ricin toxin A-chain of claim 8, wherein thevaline at amino acid residue 76 as set forth in SEQ ID NO:1 issubstituted with an isoleucine, phenylalanine, cysteine, methionine,alanine, glycine, threonine, tryptophan, tyrosine, proline, histidine,glutamic acid, glutamine, aspartic acid, asparagine, lysine, orarginine.
 10. The modified ricin toxin A-chain of claim 9, wherein thevaline at amino acid residue 76 as set forth in SEQ ID NO:1 issubstituted with threonine.